Immunotherapeutic vaccine and antibody combination therapy

ABSTRACT

The present invention relates to a combination product, composition(s) and kit of parts comprising at least (i) a therapeutic vaccine and (ii) one or more immune checkpoint modulator(s). The present invention also concerns a method for treating a proliferative or an infectious disease as well as a method for eliciting or stimulating and/or re-orienting an immune response, wherein said methods comprise administering to a subject in need thereof said combination product or said composition(s).

TECHNICAL FIELD OF THE INVENTION

The present invention generally relates to novel combinations comprisingof one or more immune checkpoint modulator(s) and at least a therapeuticvaccine (more specifically a vectorized vaccine encoding antigen(s)).Embodiments also include compositions and kits comprising suchcomponents as well as methods for treating, preventing or inhibitingproliferative and infectious diseases. The invention is of very specialinterest in the field of immunotherapy, specifically for enhancinghost's immune response and in particular for disrupting immunetolerance.

BACKGROUND ART

Using the host's immune system to eradicate persistent infectiousorganisms and malignant cells is a promising approach. This specifictype of vaccine strategy is generally referred to as immunotherapy.Widely used in traditional vaccination, immunotherapy has shown somepromise results in therapy for treating severe, chronic andlife-threatening diseases.

Numerous research groups have investigated immunotherapy as a potentialmodality for treating cancer in an attempt to stimulate the immunesystem and thus reject and destroy tumors. A vast number ofimmunotherapeutic treatments have been described in the literature fordecades using various approaches, for example cancer cells, parts ofcells, purified antigens and vectorized antigens. Cell vaccines aregenerally made up of cancer cells that have been removed from thepatient during surgery and altered in lab to make them more amenable tobe attacked by the patient's immune system before being reintroduced inthe patient. Alternatively, one may use immune cells obtained from thepatient's blood that have been exposed to cancer cells or associatedantigens, cultured in the presence of chemokines that turn them intodendritic cells before being given back to the patient by intravenousinfusion in order to help other immune system cells to attack the cancercells. The dendritic cell-based vaccine Provenge® (sipuleucel-T), wastested in advanced clinical trials to treat advanced prostate cancer andreceived FDA approval in 2010. However, such cell-based vaccines requireto be made individually for each patient and the process used to producethem is thus complex and expensive.

Antigen vaccines are made up of only one or a few protein or peptideantigen(s) that are specific for a certain type of cancers or pathogens.Once administered, they will be able to induce specific immunologicalresponses against these antigens and boost the patient's immune system.Several candidate peptide vaccines reached clinical development. Forexample, the liposomal vaccine Stimuvax® incorporates lipopeptidesgenerated from the mucin 1 (MUC1) glycoprotein that is widely expressedby common cancers. Although it did not provide in clinical trials asignificant improvement in overall survival in patients with advancednon-small cell lung cancer (NSCLC), effects were nevertheless seen insome subgroups of patients.

Vector-based vaccines have shown great promise and play an importantrole in the development of new therapeutic strategies. Vectors are usedto deliver the targeted antigen into the body. Typically, vectorsoriginate from virus, bacteria, yeast cells, or other structures thathave been altered to make them no longer harmful for the patient (e.g.inactivated, attenuated, etc). The ideal viral vector should be safe andenable efficient antigen presentation of the encoded antigens to theimmune system. Furthermore, the vector system must meet criteria thatenable its production on a large-scale basis. Live viral vectors areattractive for their ability to both express antigens from a variety ofpathogens and tumoral tissue and to facilitate antigen presentationthrough the endogenous pathway which has been shown to be important forefficient induction of cellular immune responses (Reyes-Sandoval, 2007,Immunology 121(2): 158-65). Several viral vectors have thus emerged todate, all of them having relative advantages and limits depending on theproposed application (see for example Harrop and Carroll, 2006, FrontBiosci., 11, 804-817; Inchauspé et al., 2009, Int Rev Immunol 28(1):7-19; Torresi et al., 2011, J. Hepatol. 54(6): 1273-85). Significantresearch efforts have also been undertaken to develop antigen-coding DNAplasmids and associated delivery device to stimulate protective immuneresponses (e.g. see Reyes-Sandoval and Ertl, 2001, Curr Mol Med 1(2):217-43).

For example, replication-defective adenovirus (Ad) vectors have beenextensively used because Ad infects replicating and non-replicatingcells, has a broad tissue tropism, propagates efficiently in suitablepackaging cell lines and production process is scalable and affordable(Boukhebza et al., 2014, Vaccine 32(26): 3256-63). The attenuatednon-replicative vaccinia virus Ankara strain (MVA) is also an attractivecandidate since it has been shown to induce robust cellular immuneresponses with an excellent safety profile both in the cancer andinfectious diseases fields (Boukhebza et al., 2012, Hum VaccinImmunother 8(12): 1746-57; Habersetzer et al., 2011, Gastroenterology141(3): 890-99; Fournillier et al., 2007, Vaccine 25(42): 7339-53;Drexler et al., 2004, Curr Opin Biotechnol 15(6): 506-12). MVA has beenattenuated by more than 570 passages in chicken embryo fibroblastsresulting in the loss of 15% of its genome. Consequently, MVA is unableto produce mature virions in most mammalian cells that result in areduced risk of dissemination and an increased immunogenicity due to theloss of several anti-immune defense genes (Sutter et al., 1994, Vaccine12(11): 1032-40).

However, there are limits on the immune system's ability to fightchronic infectious diseases and cancers. Sometimes the immune systemdoesn't detect the cancer or infected cells as foreign because the cellsare not different enough from normal cells. In other cases, the responsemight not be strong enough to destroy the diseased cells, especially inimmuno-compromised patients. Finally, the immune system might beineffective due to the fact that diseased cells have evolved differentways of eluding the immune system.

One of the major mechanisms of immune suppression is a process known as“T-cell exhaustion”, which results from chronic exposure to antigens andis characterized by the upregulation of inhibitory receptors. Theseinhibitory receptors serve as immune checkpoints in order to preventuncontrolled immune reactions. Various immune checkpoints acting atdifferent levels of T cell immunity have been described in theliterature, including programmed cell death protein 1 (PD-1), cytotoxicT-lymphocyte associated protein-4 (CTLA-4), Lymphocyte-activation gene 3(LAG3), B and T lymphocyte attenuator, T-cell immunoglobulin, mucindomain-containing protein 3 (TIM-3), and V-domain immunoglobulinsuppressor of T cell activation (VISTA). It has also been reported thatthe interaction of PD-1 with its ligands PDL-1 and PDL-2 plays acritical role in T cell exhaustion (Maier et al., 2007, J. Immunol. 178:2714-20; Tzeng et al., 2012, PLoS One 7: e39179).

Whatever the mechanism of action, these immune checkpoints can inhibitthe development of an efficient immune response. There is an increasinginterest of blocking such immune checkpoints as a means of inhibitingimmune system tolerance and thus rescue exhausted T cells (Leach et al.,1996, Science 271: 1734-6). A vast number of antagonistic antibodieshave been developed during the last decade (e.g. anti LAG3, -PD-L1,-CTLA-4, -PD1, etc) and three are already marketed. The first to reachthe market was the monoclonal-CTLA-4-specific antibody ipilimumab(Yervoy trade name, Bristol-Myers Squibb (BMS)) that has been approvedfor unresectable or metastatic melanoma. BMS reported that from 1861melanoma patients treated with ipilimumab 22% and 17% are still alive 3and 7 years later, respectively. Anti-PD1 nivolumab antibody (Opdivotrade name, BMS) was approved in Japan in July 2014 for malignantmelanoma. Interim phase II data showed a 32% response rate in pretreatedmetastatic melanoma with lower high grade adverse events thanIpilimumab. Anti-PD-1 pembrolizumab (Keytruda trade name, Merck), gainedaccelerated FDA approval for the treatment of unresectable or metastaticmelanoma. The marketed antibodies are also in clinical trials for otherindications including NSCLC (non-small cell lung cancer) as well as avariety of other immune checkpoint inhibitors targeting PD-1 (e.g.pidilizumab, CureTech), CTLA-4 (e.g. Tremelimumab (AstraZeneca), PD-L1(e.g. MPDL3280A, Roche), KIR (lirilumab, BMS), IDO1 (e.g. indoximod,NewLink genetics) and others.

Preclinical studies with antagonist antibodies are also pursuing ininfectious disease field (see e.g. Barber et al., 2006, Nature 439:682-7; Cecchinato et al., 2008, J. Immunol 180: 5439-47) andcombinations with different vector platforms (DNA, MVA, lentivirus,vaccinia, etc.) were envisaged. In particular, the combination of PD-L1blockade with a vaccinia virus expressing LMCV (LymphocyticChronomeningitis Virus) epitope was shown to improve the function ofepitope-specific CD8+ T cells during persistent viral infection (Ha etal., 2008, JEM 205: 543-55). Administration of anti-PD-1 antibodiestogether with a SIV gag adenovirus vector in naive macaques causedincreased in Gag-specific T cells (Finnefrock et al., 2009, J. Immunol.182: 980-7). WO2004/058801 relates to the treatment of cancer using arecombinant MVA vector encoding p53 oncogenic polypeptide in combinationwith anti-CTLA4 antibodies and CpG oligodeoxynucleotideimmunomodulators.

One may expect that cancer and infectious diseases will continue to be aserious global health threat for many years. Although availability ofantibiotics and vaccines, infectious diseases cause 100.000 deaths peryear throughout the world (data WHO 2002). On the other hand, malignantand especially metastatic tumors are often resistant to conventionaltherapies explaining the significant morbidity of some cancers. Theabove-description clearly illustrate that designing effective therapiesis a difficult task due to the numerous mechanisms set up by the host'sbody to escape immune effector cells.

SUMMARY OF THE INVENTION

In the context of the invention, the inventors identified a combinationproduct able to potentiate the patient's immune responses and/or restoreexhausted T cell-mediated immunity. Essential elements of such acombination product are a therapeutic vaccine and an immune checkpointinhibitor. The inventors surprisingly found that administrations of aMVA vector encoding a model antigen (βGal) in combination withanti-CTLA4 or anti-PD-1 antibody are surprisingly effective to reducethe volume of tumors implanted in a human cancer animal model andincrease the survival rate of those animals. The ability of suchcombinations to provide antitumor effects is a good indication that thepresent invention can be useful for treating human subjects against avariety of diseases, and especially infectious and proliferativediseases.

In a first aspect the invention provides a combination productcomprising at least a therapeutic vaccine and one or more immunecheckpoint modulator(s). Preferably, the therapeutic vaccine comprises aviral vector and more preferably a recombinant viral vector encoding anantigenic polypeptide. Preferably the immune checkpoint modulator is amonoclonal antibody capable of antagonizing at least partially theactivity of immune checkpoint such as CTLA-4 or PD-1.

The present invention also provides a composition comprising thetherapeutic vaccine and the one or more immune checkpoint modulator(s)as well as the use or method of treatment using such a composition orcombination, especially for treating infectious and proliferativediseases such as cancer.

Other and further aspects, features and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention. These embodiments aregiven for the purpose of disclosure.

DETAILED DESCRIPTION OF THE INVENTION

As described above, the inventors identified a combination productcomprising at least (i) a therapeutic vaccine and (ii) one or moreimmune checkpoint modulator(s).

As used throughout the entire application, the terms “a” and “an” areused in the sense that they mean “at least one”, “at least a first”,“one or more” or “a plurality” of the referenced components or steps,unless the context clearly dictates otherwise. For example, the term “atherapeutic vaccine” includes a plurality of therapeutic vaccines,including mixtures thereof.

The term “one or more” refers to either one or a number above one (e.g.2, 3, 4, 5, etc).

The term “and/or” wherever used herein includes the meaning of “and”,“or” and “all or any other combination of the elements connected by saidterm”.

The term “about” or “approximately” as used herein means within 10%,preferably within 8%, and more preferably within 5% of a given value orrange.

As used herein, when used to define products, compositions and methods,the term “comprising” (and any form of comprising, such as “comprise”and “comprises”), “having” (and any form of having, such as “have” and“has”), “including” (and any form of including, such as “includes” and“include”) or “containing” (and any form of containing, such as“contains” and “contain”) are open-ended and do not exclude additional,unrecited elements or method steps. Thus, a polypeptide “comprises” anamino acid sequence when the amino acid sequence might be part of thefinal amino acid sequence of the polypeptide. “Consisting essentiallyof” means excluding other components or steps of any essentialsignificance. Thus, a composition consisting essentially of the recitedcomponents would not exclude trace contaminants and pharmaceuticallyacceptable carriers. A polypeptide “consists essentially of” an aminoacid sequence when such an amino acid sequence is present withoptionally only a few additional amino acid residues. “Consisting of”means excluding more than trace elements of other components or steps.For example, a polypeptide “consists of” an amino acid sequence when thepolypeptide does not contain any amino acids but the recited amino acidsequence.

The terms “polypeptide”, “peptide” and “protein” refer to polymers ofamino acid residues which comprise at least nine or more amino acidsbonded via peptide bonds. The polymer can be linear, branched or cyclicand may comprise naturally occurring and/or amino acid analogs and itmay be interrupted by non-amino acids. As a general indication, if theamino acid polymer is more than 50 amino acid residues, it is preferablyreferred to as a polypeptide or a protein whereas if it is 50 aminoacids long or less, it is referred to as a “peptide”.

Within the context of the present invention, the terms “nucleic acid”,“nucleic acid molecule”, “polynucleotide” and “nucleotide sequence” areused interchangeably and define a polymer of any length of eitherpolydeoxyribonucleotides (DNA) (e.g. cDNA, genomic DNA, plasmids,vectors, viral genomes, isolated DNA, probes, primers and any mixturethereof) or polyribonucleotides (RNA) (e.g. mRNA, antisense RNA, SiRNA)or mixed polyribo-polydeoxyribonucleotides. They encompass single ordouble-stranded, linear or circular, natural or synthetic, modified orunmodified polynucleotides. Moreover, a polynucleotide may comprisenon-naturally occurring nucleotides and may be interrupted bynon-nucleotide components.

The term “analog”, “mutant” or “variant” as used herein refers to acomponent (polypeptide or nucleic acid) exhibiting one or moremodification(s) with respect to its native counterpart. Anymodification(s) can be envisaged, including substitution, insertionand/or deletion of one or more nucleotide/amino acid residue(s). Whenseveral mutations are contemplated, they can concern consecutiveresidues and/or non-consecutive residues. Mutation(s) can be generatedby a number of ways known to those skilled in the art, such assite-directed mutagenesis (e.g. using the Sculptor™ in vitro mutagenesissystem of Amersham, Les Ullis, France), PCR mutagenesis, DNA shufflingand by chemical synthetic techniques (e.g. resulting in a syntheticnucleic acid molecule). Preferred are analogs that retain a degree ofsequence identity of at least 80%, preferably at least 85%, morepreferably at least 90%, and even more preferably at least 98% identitywith the sequence of the native counterpart.

In a general manner, the term “identity” refers to an amino acid toamino acid or nucleotide to nucleotide correspondence between twopolypeptide or nucleic acid sequences. The percentage of identitybetween two sequences is a function of the number of identical positionsshared by the sequences, taking into account the number of gaps whichneed to be introduced for optimal global alignment and the length ofeach gap. Various computer programs and mathematical algorithms areavailable in the art to determine the percentage of identity betweenamino acid sequences, such as for example the algorithm of Needleman etWunsch. J. Mol. Biol. 48, 443-453, 1970, the Blast program available atNCBI or ALIGN in Atlas of Protein Sequence and Structure (Dayhoffed,1981, Suppl., 3: 482-9) or the needle software available from ebi.ac.ukworld wide under the name <<Align>>. Programs for determining identitybetween nucleotide sequences are also available in specialized data base(e.g. Genbank, the Wisconsin Sequence Analysis Package, BESTFIT, FASTAand GAP programs). For illustrative purposes, “at least 80% identity”means 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

As used herein, the term “isolated” refers to a component (e.g. apolypeptide, peptide, polynucleotide, vector, etc.), that is removedfrom its natural environment (i.e. separated from at least one othercomponent(s) with which it is naturally associated or found in nature).An isolated component refers to a component that is maintained in aheterologous context or purified (partially or substantially). Forexample, a nucleic acid molecule is isolated when it is separated ofsequences normally associated with it in nature (e.g. dissociated from achromosome or a genome) but it can be associated with heterologoussequences (e.g. within a recombinant vector).

The term “obtained from”, “originating” or “originate” is used toidentify the original source of a component (e.g. a polypeptide,peptide, polynucleotide, vector, etc.) but is not meant to limit themethod by which the component is made which can be, for example, bychemical synthesis or recombinant means.

The term “combination” as used herein refers to any arrangement possibleof two or more entities (e.g. at least the therapeutic vaccine and theone or more immune checkpoint modulator(s) described herein).

A “therapeutic vaccine” as used herein refers to any substance (e.g.polypeptide, polysaccharide, nucleic acid, etc.), including complexsubstance (e.g. cells, cell mixtures, live or dead organisms such asbacteria, viruses, and other microorganisms, etc. . . . ), part thereof(e.g. immunogenic fragment, epitope, cell wall, flagella, etc) oranalogs thereof, that is capable of being the target of an immuneresponse. The term encompasses “antigens” (i.e. native antigens as wellas fragments and analogs thereof).

The term “immune checkpoint modulator” refers to a molecule capable ofmodulating the function of an immune checkpoint protein in a positive ornegative way (in particular the interaction between an antigenpresenting cell (APC) and an immune T effector cell).

The term “subject” generally refers to an organism for whom any productand method of the invention is needed or may be beneficial. Typically,the organism is a mammal, particularly a mammal selected from the groupconsisting of domestic animals, farm animals, sport animals, andprimates. Preferably, the subject is a human who have been diagnosed asbeing or at risk of having a pathological condition such as aninfectious disease caused by or associated with a pathogenic organism ora proliferative disease such as cancer. The terms “subject” and“patients” may be used interchangeably when referring to a humanorganism and encompasses male and female. The subject to be treated maybe a newborn, an infant, a young adult or an adult.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect the present invention provides a combination productcomprising at least (i) a therapeutic vaccine and (ii) one or moreimmune checkpoint modulator(s).

Combination Product

In the context of the present invention, such an arrangement includes amixture of the individual entities (e.g. a single composition) meaningthat the individual entities making up the combination are placedtogether in a common container before administration to the subject. Bycontrast, distinct combinations refer to the case where the individualentities are not mixed together meaning that they are into separatecontainers (e.g. distinct compositions) for administration inconjunction with one another either concomitantly, sequentially or in aninterspersed manner.

Exemplary combinations include, but are not limited to, combination ofpolypeptides (e.g. peptide or protein-based therapeutic vaccine and oneor more immune checkpoint modulator(s) in the form of recombinantpolypeptide) or combination of nucleic acid molecule(s) (e.g. avectorized therapeutic vaccine and one or more vector(s) engineered forencoding and expressing immune checkpoint modulator(s)) as well ascombination of both polypeptide(s) and nucleic acid molecule(s) (e.g. avectorized therapeutic vaccine and one or more recombinant immunecheckpoint modulator polypeptide(s)). For illustrative purposes, asingle composition can be in the form of a) a mixture of the therapeuticvaccine with one or more immune checkpoint modulator polypeptide(s); b)a mixture of the therapeutic vaccine with vector(s) encoding of the oneor more immune checkpoint modulator(s) or c) a specific design (e.g. thetherapeutic vaccine encodes both polypeptide(s) of therapeutic interestand the one or more immune checkpoint modulator(s)). The presentinvention encompasses combinations comprising equal molar concentrationsof each entity as well as combinations with very differentconcentrations of the different entities. It is appreciated that optimalconcentration of each entity of the combination can be determined by theartisan skilled in the art. Preferably, the combination is synergisticproviding higher efficacy (e.g. an increased survival) than each entityalone.

In the context of the invention, the combination product of the presentinvention can be used for prophylaxis (e.g. to reduce the risk of havinga given disease or pathological condition) and/or therapy (e.g. in asubject diagnosed as having a given disease or pathological condition).Therapeutic use is preferred.

Therapeutic Vaccine

In one embodiment, the “therapeutic vaccine” as used herein is abiological product designed to elicit or increase immunity to aparticular target (e.g. cancerous or infected cells, etc.) through thepresence or expression of a polypeptide of interest which is expected tocause a beneficial effect on the course or a symptom of the disease orpathological condition when administered appropriately to a subject.

Several types of therapeutic vaccines can be used in the context of theinvention including, but not limited to, cell-based vaccines, peptide orpolypeptide-based vaccines and vector-based vaccines.

Representative examples of cell based vaccines can be obtained forexample

-   -   From stem cells (such as SL-701 developed by Stemline        Therapeutics for treating glioblastoma);    -   From specialized cells such as immune cells that are        reprogrammed in vitro to attack cancer cells (e.g. the Dendritic        cell vaccine developed by Immunocellular Therapeutics targeting        six tumor-associated antigens (TAA) involved in glioblastoma,        and Provenge vaccine approved for treating advanced prostate        cancer);    -   From patient's cancer cells altered in lab to make them more        amenable to be attacked by the patient's immune system; or    -   From microorganisms that have been engineered for being        avirulent or attenuated by disabling their virulent properties        and optionally for expressing polypeptides of interest.        Well-known examples of such microorganisms include without        limitation bacterium (e.g. Mycobacterium; Lactobacillus (e.g.        Lactococcus lactis); Listeria (e.g. Listeria monocytogenes)        Salmonella and Pseudomona), and yeast (e.g. Saccharomyces        cerevisiae, Schizosaccharomyces pombe, Pichia pastoris).        Representative examples of bacterium and yeast therapeutic        vaccines include Mycobacterium bovis BCG and Tarmogens^(R)        developed by GlobeImmune made from genetically-modified yeast        that express one or more disease-associated antigens.

Typically, peptide or polypeptide vaccine includes antigenicpeptide(s)/polypeptide(s) optionally mixed to an adjuvant. One may citefor illustrative purpose Newax E75 developed by Galena and Genentech forbreast cancer.

Vector-based vaccines are preferred in the context of the invention. Theterm “vector” as used herein refers to a vehicle, preferably a nucleicacid molecule or a viral particle that contains the elements necessaryto allow delivery, propagation and/or expression of biological moleculeswithin a host cell or subject. For the purpose of the invention, thevectors may be of naturally occurring genetic sources, synthetic orartificial, or some combination of natural and artificial geneticelements. This term encompasses extrachromosomal vectors (e.g. thatremain in the cell cytosol or nucleus) and integration vectors (e.g.designed to integrate into the cell genome) as well as cloning andexpression vectors.

In one embodiment, the therapeutic vaccine comprises a recombinantplasmid or viral vector. A “plasmid vector” as used herein refers to areplicable DNA construct. Usually plasmid vectors contain selectablemarker genes that allow host cells carrying the plasmid vector to beselected for or against in the presence of a corresponding selectivedrug. A variety of positive and negative selectable marker genes areknown in the art. By way of illustration, an antibiotic resistance genecan be used as a positive selectable marker gene that allows selectionof the plasmid-containing cells in the presence of the correspondingantibiotic. Suitable plasmid vectors include, without limitation, pREP4,pCEP4 (Invitrogene), pCI (Promega), pCDM8 (Seed, 1987, Nature 329: 840),pMT2PC (Kaufman et al., 1987, EMBO J. 6: 187-95), pVAX (Invitrogen) andpgWiz (Gene Therapy System Inc; Himoudi et al., 2002, J. Virol. 76:12735-46).

The term “viral vector” or “virus” or “virions” or “therapeutic virus”as used herein refers to a nucleic acid vector that includes at leastone element of a virus genome allowing packaging into a viral particle.In the context of the present invention, these terms have to beunderstood broadly as including nucleic acid vector (e.g. vector DNA) aswell as viral particles generated thereof, and especially infectiousviral particles. The term “infectious” refers to the ability of a viralvector to infect and enter into a host cell or subject.

Viral vectors can be replication-competent or selective (e.g. engineeredto replicate better or selectively in specific host cells), or can begenetically disabled so as to be replication-defective orreplication-impaired. In a preferred embodiment, the therapeutic vaccinecomprised in the combination of the invention is a replication-defectiveor replication-impaired viral vector which means that it cannotreplicate to any significant extent in normal cells, especially innormal human cells. The impairment or defectiveness of replicationfunctions can be evaluated by conventional means, such as by measuringDNA synthesis and/or viral titer in non-permissive cells. The viralvector can be rendered replication-defective by partial or totaldeletion or inactivation of regions critical to viral replication. Suchreplication-defective or impaired viral vectors typically require forpropagation, permissive cell lines which bring up or complement themissing/impaired functions.

Viral vectors can be engineered from a variety of viruses and inparticular from the group of viruses consisting of adenovirus,adenovirus-associated virus (AAV), poxvirus, herpes virus, measlesvirus, foamy virus, alphavirus, vesicular stomatis virus, Newcastledisease virus, picorna virus, Sindi virus, etc). One may use eitherparental strains as well as derivatives thereof (i.e. a virus that ismodified compared to a parental strain of said virus, e.g. bytruncation, deletion, substitution, and/or insertion of one or morenucleotide(s) contiguous or not within the viral genome).Modification(s) can be within endogenous viral genes (e.g. coding and/orregulatory sequences) and/or within intergenic regions. Moreover,modification(s) can be silent or not (e.g. resulting in a modified viralgene product). Modification(s) can be made in a number of ways known tothose skilled in the art using conventional molecular biologytechniques.

Preferably, the modifications encompassed by the present inventionaffect, for example, virulence, toxicity, or pathogenicity of the viruscompared to a virus without such modification, but do not completelyinhibit infection and production of new virus at least in permissivecells. Said modification(s) preferably lead(s) to the synthesis of adefective protein (or lack of synthesis) so as to be unable to ensurethe activity of the protein produced under normal conditions by theunmodified gene. Exemplary modifications are disclosed in the literaturewith a specific preference for those altering viral genes involved inDNA metabolism, host virulence and IFN pathway (see e.g. Guse et al.,2011, Expert Opinion Biol. Ther. 11(5):595-608). Other suitablemodifications include the insertion of exogenous gene(s) (e.g. nucleicacid molecule(s) of interest) as described hereinafter.

A particularly suitable viral vector to be comprised in the therapeuticvaccine in use herein is obtained from a poxvirus. As used herein theterm “poxvirus” refers to a virus belonging to the Poxviridae familywith a preference for the Chordopoxvirinae subfamily directed tovertebrate host which includes several genus such as Orthopoxvirus,Capripoxvirus, Avipoxvirus, Parapoxvirus, Leporipoxvirus andSuipoxvirus. Orthopoxviruses are preferred in the context of the presentinvention as well as the Avipoxviruses including Canarypoxvirus (e.g.ALVAC) and Fowlpoxvirus (e.g. the FP9 vector).

In a preferred embodiment, the therapeutic vaccine comprises a poxviralvector belonging to the Orthopoxvirus genus and even more preferably tothe vaccinia virus (VV) species. Vaccinia viruses are large, complex,enveloped viruses with a linear, double-stranded DNA genome ofapproximately 200 kb in length which encodes numerous viral enzymes andfactors that enable the virus to replicate independently from the hostcell machinery. Two distinct infectious viral particles exist, theintracellular IMV (for intracellular mature virion) surrounded by asingle lipid envelop that remains in the cytosol of infected cells untillysis and the double enveloped EEV (for extracellular enveloped virion)that buds out from the infected cell. Any vaccinia virus strain can beused in the context of the present invention including, withoutlimitation, Western Reserve (WR), Copenhagen (Cop), Lister, LIVP, Wyeth,Tashkent, Tian Tan, Brighton, Ankara, MVA (Modified vaccinia virusAnkara), LC16M8, LC16M0 strains, etc. and any derivative thereof.

Engineered poxviruses can be used with modifications aimed at improvingsafety (e.g. increased attenuation) and/or efficacy (e.g. improvedselectivity for cancer cells and/or decreasing toxicity in healthycells) of the resulting virus. One may cite more particularly defectivemodifications within the thymidine kinase (J2R; see Weir and Moss, 1983,Genbank accession number AAA48082), the deoxyuridine triphosphatase(F2L), the viral hemagglutinin (A56R); the small (F4L) and/or the large(I4L) subunit of the ribonucleotide reductase, the serine proteaseinhibitor (B13R/B14R) and the complement 4b binding protein (C3L).Representative examples of suitable VV for use in this invention includeNYVAC (U.S. Pat. No. 5,494,807) as well as TK-defective, TK- andF2L-defective (WO2009/065547) and TK- and I4L-defective VV(WO2009/065546). The gene nomenclature used herein is that of CopenhagenVaccinia strain. It is also used herein for the homologous genes ofother poxviridae unless otherwise indicated. However, gene nomenclaturemay be different according to the pox strain but correspondence betweenCopenhagen and other vaccinia strains are generally available in theliterature.

Sequences of the genome of various Poxviridae, are available in the artin specialized databanks such as Genebank. For example, the vacciniavirus strains Western Reserve, Copenhagen, Cowpoxvirus andCanarypoxvirus genomes are available in Genbank under accession numbersNC_006998, M35027, NC_003663, NC_005309, respectively. A particularlyappropriate viral vector in the context of the present invention is MVAdue to its highly attenuated phenotype (Mayr et al., 1975, Infection 3:6-14; Sutter and Moss, 1992, Proc. Natl. Acad. Sci. USA 89: 10847-51), amore pronounced IFN-type 1 response generated upon infection compared tonon-attenuated vectors and availability of the sequence of its genome inthe literature (Antoine et al., 1998, Virol. 244: 365-96) and in Genbank(under accession number U94848).

Other viral vectors appropriate in the context of the invention aremorbillivirus which can be obtained from the paramyxoviridae family,with a specific preference for measles virus. Various attenuated strainsare available in the art (Brandler et al, 2008, CIMID, 31: 271; Singh etal., 1999, J. Virol. 73(6): 4823), such as and without limitation, theEdmonston A and B strains (Griffin et al., 2001, Field's in Virology,1401-1441), the Schwarz strain (Schwarz A, 1962, Am J Dis Child, 103:216), the S-191 or C-47 strains (Zhang et al., 2009, J Med Virol. 81(8): 1477). One may also use recombinant Newcastle Disease Virus (NDV)(Bukreyev and Collins, 2008, Curr Opin Mol Ther 10: 46-55) with aspecific preference for an attenuated strain thereof such as MTH-68 thatwas already used in cancer patients (Csatary et al., 1999, Anti CancerRes 19: 635-8) and NDV-HUJ, which showed promising results inglioblastoma patients (isracast.com Mar. 1, 2006).

Still another suitable viral vector for use in the present invention isan adenoviral vector. It can be derived from a variety of human oranimal adenoviruses (e.g. canine, ovine, simian, etc) and any serotypecan be employed. It can also be a chimeric adenovirus (WO2005/001103).One of skill will recognize that elements derived from multipleserotypes can be combined in a single adenovirus.

Desirably, the adenoviral vector originates from a human Ad, includingthose of rare serotypes, or from a primate (e.g. chimpanzee, gorilla).Representative examples of human adenoviruses include subgenus C (e.g.Ad2 Ad5 and Ad6), subgenus B (e.g. Ad3, Ad7, Ad11, Ad14, Ad34, Ad35 andAd50), subgenus D (e.g. Ad19, Ad24, Ad26, Ad48 and Ad49) and subgenus E(Ad4). Representative examples of chimp Ad include without limitationAdCh3 (Peruzzi et al., 2009, Vaccine 27: 1293-300) and AdCh63 (Dudarevaet al, 2009, Vaccine 27: 3501-4) and any of those described in the art(see for example, WO2010/086189; WO2009/105084; WO2009/073104;WO2009/073103; WO2005/071093; and WO03/046124). A number of adenovirusesare now well characterized genetically and biochemically (Hoffmann etal., 2007, Human Gene Ther. 18: 51-62). An exemplary genome sequence ofhuman adenovirus type 5 (Ad5) is found in GenBank Accession M73260 andin Chroboczek et al. (1992, Virol. 186: 280-5).

Preferably, the adenovirus employed in this invention isreplication-defective, e.g. by total or partial deletion of E1 region.An appropriate E1 deletion extends from approximately positions 459 to3510 by reference to the sequence of the Ad5 disclosed in the GenBankunder the accession number M 73260. Preferably, the virus retains afunctional viral pIX gene. The adenoviral genome may comprise additionalmodification(s) (e.g. deletion of all or part of other essential E2and/or E4 regions as described in WO94/28152; Lusky et al, 1998, J.Virol 72: 2022). In addition, the non-essential E3 region can also bemutated or deleted.

More preferably, the adenovirus comprised in the therapeutic vaccine ofthe invention is a human adenovirus of serotype 5 (Ad5), defective forE1 and/or E3 function and comprising a nucleic acid molecule of interestinserted in the E1 region.

The present invention also encompasses vectors or viral particlescomplexed to lipids or polymers (e.g. polyethylene glycol) to formparticulate structures such as liposomes, lipoplexes or nanoparticles aswell as vectors or viral particles modified to allow preferentialtargeting to a specific host cell. A characteristic feature of targetedvectors is the presence at their surface of a ligand capable ofrecognizing and binding to a cellular and surface-exposed component.Examples of suitable ligands include antibodies or fragments thereofdirected to cell-specific, tissue-specific and pathogen-associatedmarkers. Targeting can be carried out by genetically inserting theligand into a polypeptide present on the surface of the virus (e.g. inthe adenoviral fiber or in the poxviral p14 IMV exposed polypeptide,etc.) or by chemically modifying the viral surface envelope.Furthermore, when using a virus-based therapeutic vaccine, said viruscan be live, attenuated, inactivated or killed.

Polypeptides of Interest

The therapeutic vaccine comprised in the combination product of thepresent invention preferably contains or encodes one or morepolypeptides of therapeutic interest that can compensate forpathological symptoms, e.g. by acting through toxic effects to limit orremove harmful cells from the body, by improving immunity or byreversing immune exhaustion mechanisms. Such polypeptides may be encodedby native genes or genes obtained from the latter following suitablesequence modification(s). In the context of the invention, thepolypeptide of interest can be of mammal origin (e.g. human) or not(e.g. of bacterial or viral origin).

Advantageously, the therapeutic vaccine in use in the present inventioncomprises or encodes one or more polypeptide(s) selected from the groupconsisting of suicide gene products, immunostimulatory polypeptides andantigenic polypeptides. Preferred antigenic polypeptides for use hereinare tumor-associated antigens and antigens of pathogenic organisms.Preferably, the polypeptide of interest is not an oncogenictranscription factor such as p53.

Suicide Genes

The term “suicide gene” refers to a nucleic acid molecule coding for aprotein (e.g. enzyme) able to convert a precursor of a drug into acytotoxic compound. Appropriate suicide genes for use in this inventionare disclosed in the following table with the corresponding prodrug (ordrug precursor) and the active (cytotoxic) drug.

TABLE 1 Enzyme Prodrug Active Drug Thymidine phosphorylase 5-FU 5-FdUMP5′-DFUR 5-FU Deoxycitidine kinase Gemcitabine Gemcitabine monophosphateCytidine deaminase 5′-DFCR 5′-DFUR Cytosine deaminase 5-FC 5-FU Uracil5-FU 5-FUMP phosphoribosyltransferase Thymidine phosphorylase 5-FU5-FdUMP Thymidine kinase (HSV) Ganciclovir Ganciclovir-triphosphatenucleotide Nitroreductase CB1954 5-(Aziridin-1-yl)-4-hydroxyl-amino-2-nitro-benzamide Cytochrome P450 Ifosfamide Isophosphoramidemustard Cyclophosmamide Phosphoramide mustard Purine-nucleosideFludarabine 2-Fluoroadenine phosphorylase Alkaline phosphatase Etoposidephosphate Etoposide Mitomycin C phosphate Mitomycin C N-(4-phophonooxy-Doxorubicin phenylacetyl)doxorubicin Carboxypeptidase Methotrexate-aminoacids Methotrexate Penicillin amidase N-(phenylacetyl) doxorubicinDoxorubicin β-Lactamase C-DOX Doxorubicin

Desirably, the therapeutic vaccine comprises or encodes a polypeptidehaving at least cytosine deaminase (CDase) activity. CDase encodingnucleic acid molecule can be obtained from any prokaryotes and lowereukaryotes such as Saccharomyces cerevisiae (FCY1 gene), CandidaAlbicans (FCA1 gene) and Escherichia coli (codA gene).

Alternatively or in combination, the therapeutic vaccine comprises orencodes a polypeptide having uracil phosphoribosyl transferase (UPRTase)activity. UPRTase-encoding nucleic acid molecule can be obtained from E.coli (Andersen et al., 1992, European J. Biochem. 204: 51-56),Lactococcus lactis (Martinussen et al., 1994, J. Bacteriol. 176:6457-63), Mycobacterium bovis (Kim et al., 1997, Biochem. Mol. Biol.Internat. 41: 1117-24), Bacillus subtilis (Martinussen et al., 1995, J.Bacteriol. 177: 271-4) and yeast (e.g. S. cerevisiae FUR1 disclosed byKern et al., 1990, Gene 88: 149-57).

The nucleotide sequence of such CDase and UPRTase-encoding nucleic acidmolecules and amino acids of the encoded enzyme are available inspecialized data banks (SWISSPROT EMBL, Genbank, Medline and the like).Functional analogues may also be used. Such analogues preferably have anamino acid sequence having a degree of identity of at least 70%,advantageously of at least 80%, preferably of at least 90%, and mostpreferably of at least 95% with the native polypeptide. It is within thereach of the skilled person to engineer analogs from the published data,and test the enzymatic activity in an acellular or cellular systemaccording to conventional techniques (see e.g. EP998568). Forillustrative purposes, suitable functional analogues comprise theN-terminally truncated FUR1 mutant described in EP998568 (with adeletion of the 35 first residues up to the second Met residue presentat position 36 in the native protein) which exhibits a higher UPRTaseactivity than that of the native enzyme as well as the FCY1::FUR1fusions named FCU1 (amino acid sequence represented in the sequenceidentifier SEQ ID NO: 1 of WO2009/065546) and FCU1-8 described inWO96/16183, EP998568 and WO2005/07857.

Immunostimulatory Polypeptides

As used herein, the term “immunostimulatory polypeptide” refers to apolypeptide which has the ability to stimulate the immune system, in aspecific or non-specific way. A vast number of proteins are known in theart for their ability to exert an immunostimulatory effect. Examples ofsuitable immunostimulatory proteins in the context of the inventioninclude without limitation cytokines, with a specific preference forinterleukins (e.g. IL-2, IL-6, IL-12, IL-15, IL-24), chemokines (e.g.CXCL10, CXCL9, CXCL11), interferons (e.g. IFNα, IFNβ, IFNγ), tumornecrosis factor (TNF), colony-stimulating factors (e.g. GM-CSF, C-CSF,M-CSF . . . ), APC (for Antigen Presenting Cell)-exposed proteins (e.g.B7.1, B7.2 and the like), growth factors (Transforming Growth FactorTGF, Fibroblast Growth Factor FGF, Vascular Endothelial Growth FactorsVEGF, and the like), major histocompatibility complex (MHC) antigens ofclass I or II, apoptosis inducers or inhibitors (e.g. Bax, Bcl2, BclX .. . ) and immunotoxins. Preferably, the immunostimulatory protein is aninterleukin or a colony-stimulating factor (e.g. GM-CSF).

Antigenic Polypeptides

In one embodiment, the therapeutic vaccine contains or encodes anantigen in connection with the disease to treat. The term “antigenic”refers to the capacity of eliciting or stimulating an immune response(e.g. a cell-mediated and/or humoral immunity). The antigen stimulatesthe body's immune system to recognize the target as foreign so that theimmune system can more easily recognize and destroy it when laterencounters. The present invention encompasses native antigenicpolypeptides (present in/on live or dead organisms or cells) as well asmodified version thereof (analogs, fragments) and combination thereof asdescribed herein.

In the context of the invention, preferred antigens contained in orencoded by the therapeutic vaccine are tumour-specific or tumour-relatedantigens (i.e. tumor-associated antigens) as well as antigens formpathogenic organisms such as virus, bacteria, parasite and the like aswell as allergens.

Viral antigenic polypeptides include for example antigens from hepatitisviruses A, B, C, D and E, immunodeficiency viruses (e.g. HIV), herpesviruses, cytomegalovirus, varicella zoster, papilloma viruses, EpsteinBarr virus, influenza viruses, para-influenza viruses, adenoviruses,coxsakie viruses, picorna viruses, rotaviruses, respiratory syncytialviruses, pox viruses, rhinoviruses, rubella virus, papovirus, mumpsvirus, measles virus. Some non-limiting examples of HIV antigens includegp120 gp40, gp160, p24, gag, pol, env, vif, vpr, vpu, tat, rev, nef tat,nef. Some non-limiting examples of human herpes viruses antigens includegH, gL gM gB gC gK gE or gD or Immediate Early protein such as ICP27,ICP47, ICP4, ICP36 from HSV1 or HSV2. Some non-limiting examples ofcytomegalovirus antigens include gB. Some non-limiting examples ofderived from Epstein Barr virus (EBV) include gp350. Some non-limitingexamples of Varicella Zoster Virus antigens include gp1, 11, 111 andIE63. Some non-limiting examples of hepatitis C virus antigens includesenv E1 or E2 protein, core protein, NS2, NS3, NS4a, NS4b, NS5a, NS5b,p7. Some non-limiting examples of human papilloma viruses (HPV) antigensinclude L1, L2, E1, E2, E3, E4, E5, E6, E7. Antigens derived from otherviral pathogens, such as Respiratory Syncytial virus (e.g. F and Gproteins), parainfluenza virus, measles virus, mumps virus, flaviviruses(e.g. Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus,Japanese Encephalitis Virus) and Influenza virus cells (e.g. HA, NP, NA,or M proteins) can also be used in accordance with the presentinvention.

Bacterial antigenic polypeptides include for example antigens fromMycobacteria causing TB and leprosy, pneumocci, aerobic gram negativebacilli, mycoplasma, staphyloccocus, streptococcus, salmonellae,chlamydiae, neisseriae and the like.

Parasitic antigenic polypeptides include for example antigens frommalaria, leishmaniasis, trypanosomiasis, toxoplasmosis, schistosomiasisand filariasis.

Allergenic polypeptides refer to any substance that can induce anallergic or asthmatic response in a susceptible subject. Allergensinclude pollens, insect venoms, animal dander dust, fungal spores anddrugs (e.g. penicillin).

Tumor-associated antigenic polypeptides (TAA) include various categoriesof antigens, e.g. those which are normally silent (i.e. not expressed)in normal cells, those that are expressed only at low levels or atcertain stages of differentiation and those that are temporallyexpressed such as embryonic and foetal antigens as well as thoseresulting from mutation of cellular genes, such as oncogenes (e.g.activated ras oncogene), proto-oncogenes (e.g. ErbB family), or proteinsresulting from chromosomal translocations. Such tumor-associatedantigens also encompass antigens encoded by pathogenic organisms thatare capable to induce a malignant condition in a subject (especiallychronically infected subject) such as RNA and DNA tumor viruses (e.g.HPV, HCV, EBV, etc) and bacteria (e.g. Helicobacter pilori). Somenon-limiting examples of tumor-associated antigens include, withoutlimitation, MART-1/Melan-A, gp100, Dipeptidyl peptidase IV (DPPIV),adenosine deaminase-binding protein (ADAbp), cyclophilin b, Colorectalassociated antigen (CRC)-C017-1A/GA733, Carcinoembryonic Antigen (CEA)and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, ProstateSpecific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, andPSA-3, prostate-specific membrane antigen (PSMA), T-cellreceptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-A1,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9,MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3),MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05),GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4,GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V,MUM-1, CDK4, tyrosinase, p53, MUC family (e.g. MUC1, MUC16, etc; seee.g. U.S. Pat. No. 6,054,438; WO98/04727; or WO98/37095), HER2/neu,p21ras, RCAS1, alpha-fetoprotein, E-cadherin, alpha-catenin,beta-catenin and gamma-catenin, p120ctn, gp100.sup.Pmel117, PRAME,NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin,Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, Smadfamily of tumor antigens brain glycogen phosphorylase, SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2 andviral antigens such as the HPV-16 and HPV-18 E6 and E7 antigens and theEBV-encoded nuclear antigen (EBNA)-1 as well as markers(beta-galactosidase, luciferase . . . ).

The present invention also encompasses therapeutic vaccinecomprising/expressing two or more polypeptides of interest as describedabove, e.g. at least two antigens, at least one antigen and oneimmunostimulatory polypeptide, at least two antigens and oneimmunostimulatory polypeptide, etc.

A preferred therapeutic vaccine comprised in the combination product ofthe invention comprises or encodes one or more polypeptides of interestselected from the group consisting of:

-   -   The MUC-1 antigen    -   HPV E6 and E7 antigens, in particular non-oncogenic variants        thereof;    -   The human IL-2    -   The human GM-CSF;    -   The FCU-1 suicide gene;    -   HBV antigens, in particular HBV pol, HBsAg and/or core;    -   Mycobacteria (e.g. MtB or M bovis) antigens in particular one or        more selected from the group consisting of RpfB, RpfD, Ag85A,        Ag85B, ESAT6, CFP10, TB10.4, Rv0111, Rv0287, Rv0569, Rv1733,        Rv1813, Rv2029, Rv2626, Rv3407, Rv3477, Rv3478, and    -   The HCV NS antigens (e.g. NS3, NS4a, NS4b, NS5a, NS5b).

The present invention encompasses the use/expression of nativepolypeptide(s) of interest as well as analogs thereof (e.g. fragmentsthereof such as peptides; and modified ones), especially when the nativepolypeptide exerts undesired properties (e.g. oncogenic or transformingproperties, cytotoxicity, etc). For example, to circumvent oncogenicityof HPV E6 and E7 polypeptides, one may use or express non oncogenicanalogs displaying reduced capacity to bind p53 and Rb, respectively.Such non oncogenic analogs are described e.g. in Munger et al. (1989,EMBO J. 8: 4099-105); Crook et al. (1991, Cell 67: 547-56); Heck et al.(1992, Proc. Natl. Acad. Sci. USA 89: 4442-6); Munger et al. (1991, J.Virol. 65: 3943-8); Phelps et al. (1992, J. Virol. 66, 2418-27) andWO99/03885. A non-oncogenic HPV-16 E6 variant which is suitable for thepurpose of the present invention is deleted of one or more amino acidresidues located from approximately position 118 to approximatelyposition 122 (+1 representing the first methionine residue of the nativeHPV-16 E6 polypeptide), with a special preference for the completedeletion of residues 118 to 122 (CPEEK). A non-oncogenic HPV-16 E7variant which is suitable for the purpose of the present invention isdeleted of one or more amino acid residues located from approximatelyposition 21 to approximately position 26 (+1 representing the firstamino acid of the native HPV-16 E7 polypeptide, with a specialpreference for the complete deletion of residues 21 to 26 (DLYCYE). Inone embodiment, it might be advantageous to modify or include additionalfeatures to the polypeptide of interest so as to improve its immunogenicactivity and/or therapeutic activity. For example, it can be useful toassociate (e.g. by mixture, fusion or independent expression) in a sametherapeutic vaccine (in particular a vector-based therapeutic vaccine):

-   -   A nucleotide sequence encoding an antigen (e.g. a TAA, or an        antigen from a pathogenic organism as previously described), and    -   A nucleotide sequence encoding an immunostimulatory polypeptide        such a cytokine or an interleukin (for instance IL-2; tumour        necrosis factor (TNF); interferon (IFN); colony stimulating        factor (CSF), or granulocyte-macrophage colony-stimulating        factor (GMCSF).

One may also use or express with the polypeptide of interest one or morepeptides or polypeptides capable of enhancing immunogenicity. Suchpeptides or polypeptides have been described in the literature andinclude, without limitation, calreticulin (Cheng et al., 2001, J. Clin.Invest. 108: 669-78), Mycobacterium tuberculosis heat shock protein 70(HSP70) (Chen et al., 2000, Cancer Res. 60: 1035-42), ubiquitin(Rodriguez et al., 1997, J. Virol. 71: 8497-503), bacterial toxin suchas the translocation domain of Pseudomonas aeruginosa exotoxin A(ETA(dIII)) (Hung et al., 2001 Cancer Res. 61: 3698-703) as well asT_(H) Pan-Dr epitope (Sidney et al., 1994, Immunity 1: 751), pstS1 GCGepitope (Vordermeier et al., 1992, Eur. J. Immunol. 22: 2631), tetanustoxoid P2TT (Panina-Bordignon et al., 1989, Eur. J. Immunol. 19: 2237)and P30TT (Demotz et al., 1993, Eur. J. Immunol. 23: 425) peptides, andinfluenza epitope (Lamb et al., 1982, Nature 300: 66; Rothbard et al.,1989, Int. Immunol. 1: 479).

Other suitable structural features are those which are beneficial to thesynthesis, processing, stability and solubility of the polypeptide ofinterest that is used in or expressed by the therapeutic vaccine of theinvention; e.g. those aimed to modify potential cleavage sites,potential glycosylation sites and/or membrane anchorage so as to improveMHC class I and/or MHC class II presentation. Membrane presentation canbe achieved by incorporating in the polypeptide of interest amembrane-anchoring sequence and a secretory sequence (i.e. a signalpeptide) if the native polypeptide lacks it. Briefly, signal peptidesusually comprise 15 to 35 essentially hydrophobic amino acids which arethen removed by a specific ER (endoplasmic reticulum)-locatedendopeptidase to give the mature polypeptide. Trans-membrane peptidesare also highly hydrophobic in nature and serve to anchor thepolypeptides within cell membrane. The choice of the trans-membraneand/or signal peptides which can be used in the context of the presentinvention is vast. They may be obtained from cellular or viralpolypeptides such as those of immunoglobulins, tissue plasminogenactivator, insulin, rabies glycoprotein, the HIV virus envelopeglycoprotein or the measles virus F protein or may be synthetic.Preferably, the secretory sequence is inserted at the N-terminus of thepolypeptide downstream of the codon for initiation of translation andthe membrane-anchoring sequence at the C-terminus, preferablyimmediately upstream of the stop codon.

Recombinant Vector

In a preferred embodiment, the therapeutic vaccine comprises arecombinant vector engineered to express at least one nucleic acidmolecule encoding a polypeptide of interest as described herein. It maybe easily generated by a number of ways known to those skilled in theart (e.g. cloning, PCR amplification, DNA shuffling). For example, sucha nucleic acid molecule can be isolated independently from any availablesource (e.g. biologic materials described in the art, cDNA and genomiclibraries, viral genomes or any prior art vector known to include it)using sequence data available to the skilled person and the sequenceinformation provided herein, and then suitably cloned by conventionalmolecular biology techniques. Alternatively, they can also be generatedby chemical synthesis in automatized process (e.g. assembled fromoverlapping synthetic oligonucleotides or synthetic gene). Preferably,such a nucleic acid molecule of interest is obtained from cDNA and doesnot comprise intronic sequences. Modification(s) can be generated by anumber of ways known to those skilled in the art, such as chemicalsynthesis, site-directed mutagenesis, PCR mutagenesis, etc.

In addition, the nucleic acid molecule for use in this invention can beoptimized for providing high level expression in a particular host cellor subject. It has been indeed observed that, the codon usage patternsof organisms are highly non-random and the use of codons may be markedlydifferent between different hosts. As the therapeutic gene might be fromprokaryote (e.g. bacterial or viral antigen) or lower eukaryote (e.g.the suicide gene) origin, it may have an inappropriate codon usagepattern for efficient expression in higher eukaryotic cells (e.g.human). Typically, codon optimization is performed by replacing one ormore “native” codon corresponding to a codon infrequently used by one ormore codon encoding the same amino acid which is more frequently used inthe subject to treat. It is not necessary to replace all native codonscorresponding to infrequently used codons since increased expression canbe achieved even with partial replacement.

Further to optimization of the codon usage, expression can also beimproved through additional modifications of the nucleotide sequence.For example, the nucleic acid sequence can be modified so as to preventclustering of rare, non-optimal codons being present in concentratedareas and/or to suppress or modify “negative” sequence elements whichare expected to negatively influence expression levels. Such negativesequence elements include without limitation the regions having veryhigh (>80%) or very low (<30%) GC content; AT-rich or GC-rich sequencestretches; unstable direct or inverted repeat sequences; R A secondarystructures; and/or internal cryptic regulatory elements such as internalTATA-boxes, chi-sites, ribosome entry sites, and/or splicingdonor/acceptor sites.

Moreover, when homologous nucleic acid molecules are to be expressed,such homologous sequences can be degenerated over the full lengthnucleic acid molecule or portion(s) thereof so as to reduce sequencehomology. It is indeed advisable to degenerate the portions of nucleicacid sequences that show a high degree of sequence identity (e.g. thesame antigen obtained from various serotypes of a given virus such asHPV-16 and HPV-18 E6 and/or E7 antigens; overlapping antigens such asHBV antigens) so as to avoid homologous recombination problems duringproduction process and the skilled person is capable of identifying suchportions by sequence alignment.

The nucleic acid molecule(s) encoding the polypeptide(s) of interest maybe inserted in the vector, e.g. within a viral gene, an intergenicregion, in a non-essential gene or region or in place of viralsequences. The general conditions for constructing and producingrecombinant poxviruses are well known in the art (see for exampleWO2010/130753; WO03/008533; U.S. Pat. No. 6,998,252; U.S. Pat. No.5,972,597 and U.S. Pat. No. 6,440,422). The nucleic acid molecule(s) ofinterest is/are preferably inserted within the poxviral genome in anon-essential locus. Thymidine kinase gene is particularly appropriatefor insertion in Copenhagen vaccinia vectors and deletion II or III forinsertion in MVA vector (WO97/02355; Meyer et al., 1991, J. Gen. Virol.72: 1031-8). The general conditions for constructing and producingrecombinant measles viruses are well known in the art. Insertion of thenucleic acid molecule(s) of interest between P and M genes or between Hand L genes is particularly appropriate. The general conditions forconstructing and producing recombinant adenoviruses are well known inthe art (see e.g. Chartier et al., 1996, J. Virol. 70: 4805-10 andWO96/17070). E1 or E3 region is the preferred site of insertion for thenucleic acid molecule(s) to be expressed which can be positioned insense or antisense orientation relative to the natural transcriptionaldirection of the region in question.

In a particularly preferred embodiment, the therapeutic vaccine isselected from the group consisting of:

-   -   A MVA virus encoding the MUC-1 TAA and human IL-2 as represented        by TG4010 described in WO92/07000, U.S. Pat. No. 5,861,381 and        Limacher and Quoix (2012, Oncolmmunology 1(5): 791-2);    -   A MVA virus encoding a fusion of NS3 and NS4 HCV antigens and        NS5b antigen (as represented by TG4040 described in        WO2004/111082);    -   A MVA virus encoding membrane anchored HPV-16 non-oncogenic E6        and E7 antigens and human IL-2 as represented by TG4001        described in WO99/03885;    -   A MVA virus encoding the FCU1 gene as represented by TG4023        (WO99/54481); and    -   A MVA virus encoding a combination of TB antigens (as described        in WO2014/009438).

Expression of the Nucleic Acid Molecule(s) Encoding the Polypeptide(s)of Interest

In accordance with the present invention, the nucleic acid molecule(s)expressed by the therapeutic vaccine for use in the invention is/areoperably linked to suitable regulatory elements for expression in adesired host cell or subject.

As used herein, the term “regulatory elements” or “regulatory sequence”refers to any element that allows, contributes or modulates theexpression of the nucleic acid molecule(s) in a given host cell orsubject, including replication, duplication, transcription, splicing,translation, stability and/or transport of the nucleic acid(s) or itsderivative (i.e. m RNA). As used herein, “operably linked” means thatthe elements being linked are arranged so that they function in concertfor their intended purposes. For example, a promoter is operably linkedto a nucleic acid molecule if the promoter effects transcription fromthe transcription initiation to the terminator of said nucleic acidmolecule in a permissive host cell.

It will be appreciated by those skilled in the art that the choice ofthe regulatory sequences can depend on factors such as the nucleic acidmolecule(s) itself, the vector from which it is expressed, the level ofexpression desired, etc. The promoter is of special importance. In thecontext of the invention, it can be constitutive directing expression ofthe nucleic acid molecule(s) in many types of cells or specific tocertain types of cells or tissues or regulated in response to specificevents or exogenous factors (e.g. by temperature, nutrient additive,hormone, etc) or according to the phase of a viral cycle (e.g. late orearly). One may also use promoters that are repressed during theproduction step in response to specific events or exogenous factors, inorder to optimize production of the therapeutic vaccine and circumventpotential toxicity of the expressed polypeptide(s).

Suitable constitutive promoters for expression in recombinant adenovirusand plasmid vectors include, but are not limited to, the cytomegalovirus(CMV) immediate early promoter (U.S. Pat. No. 5,168,062), the RSVpromoter, the adenovirus major late promoter, the phosphoglycero kinase(PGK) promoter (Adra et al., 1987, Gene 60: 65-74), the thymidine kinase(TK) promoter of herpes simplex virus (HSV)-1 and the T7 polymerasepromoter (WO98/10088). Vaccinia virus promoters are particularly adaptedfor expression in recombinant poxviruses. Representative examplesinclude without limitation the vaccinia 7.5K, HSR, 11K7.5 (Erbs et al.,2008, Cancer Gene Ther. 15(1): 18-28), TK, pB2R, p28, p11 and K1Lpromoter, as well as synthetic promoters such as those described inChakrabarti et al. (1997, Biotechniques 23: 1094-7; Hammond et al, 1997,J. Virol Methods 66: 135-8; and Kumar and Boyle, 1990, Virology 179:151-8) as well as early/late chimeric promoters. Promoters suitable formeasles viruses include without limitation any promoter directingexpression of measles transcription units (Brandler and Tangy, 2008,CIMID 31: 271).

Those skilled in the art will appreciate that the regulatory elementscontrolling the expression of the nucleic acid molecule(s) of interestmay further comprise additional elements for proper initiation,regulation and/or termination of transcription (e.g. polyA transcriptiontermination sequences), mRNA transport (e.g. nuclear localization signalsequences), processing (e.g. splicing signals), and stability (e.g.introns and non-coding 5′ and 3′ sequences), translation (e.g. aninitiator Met, tripartite leader sequences, IRES ribosome binding sites,signal peptides, etc.) and purification steps (e.g. a tag). In apreferred embodiment, the therapeutic vaccine for use in the inventioncomprises a MVA vector which contains inserted into its genome(preferably in deletion II) a nucleic acid molecule encoding atumor-associated antigen such as MUC-1 (preferably under thetranscriptional control of the early/late vaccinia pH5R promoter) and anucleic acid molecule encoding an immunostimulatory polypeptide such asthe human IL-2 (preferably under the transcriptional control of theearly/late vaccinia p7.5 promoter). More preferably, the encoded MUC1antigen comprises an amino acid sequence that is at least 90% identicalto SEQ ID NO: 1. Even more preferably, the MUC1 antigen is encoded by anucleotide sequence that is at least 80% identical to SEQ ID NO: 2.

Production of Virus-Based Therapeutic Vaccine

In a preferred embodiment, the therapeutic vaccine present in thecombination product of the present invention is a viral vector.Typically, viral vectors are produced into a suitable host cell lineusing conventional techniques including a) preparing a producer (e.g.permissive) host cell, b) transfecting or infecting the preparedproducer host cells, c) culturing the transfected or infected host cellunder suitable conditions so as to allow the production of the vector(e.g. infectious viral particles), d) recovering the produced vectorfrom the culture of said cell and optionally e) purifying said recoveredvector.

As used herein, the term “host cell” should be understood broadlywithout any limitation concerning particular organization in tissue,organ, or isolated cells. Such cells may be of a unique type of cells ora group of different types of cells such as cultured cell lines, primarycells and dividing cells. In the context of the invention, the term“host cells” include prokaryotic cells, lower eukaryotic cells such asyeast, and other eukaryotic cells such as insect cells, plant andmammalian (e.g. human or non-human) cells as well as producer cellscapable of producing the plasmid or virus-based therapeutic vaccineand/or the immune checkpoint modulator(s) for use in the invention. Thisterm also includes cells which can be or has been the recipient of thecombination product described herein as well as progeny of such cells.

In step a), suitable producer cells depend on the type of viral vectorto be amplified. Replication-defective recombinant adenoviruses aretypically propagated and produced in a cell that supplies in trans theadenoviral protein(s) encoded by those genes that have been deleted orinactivated in the replication-defective adenovirus, thus allowing thevirus to replicate in the cell. Suitable cell lines for complementingE1-deleted adenoviruses include the HEK-293 cells (Graham et al., 1997,J. Gen. Virol. 36: 59-72) as well as the HER-96 and PER-C6 cells (e.g.Fallaux et al., 1998, Human Gene Ther. 9: 1909-1917; WO97/00326) and E1A549 (Imler et al., 1996, Gene Ther. 3: 75-84) or any derivative ofthese cell lines. But any other cell line described in the art can alsobe used in the context of the present invention, especially cell linesapproved for producing products for human use. The infectious adenoviralparticles may be recovered from the culture supernatant and/or from thecells after lysis. They can be further purified according to standardtechniques (ultracentrifugation in a cesium chloride gradient,chromatography, etc. as described for example in WO96/27677, WO98/00524,WO98/22588, WO98/26048, WO00/40702, EP1016711 and WO00/50573).

MVA is strictly host-restricted and is typically amplified on aviancells, either primary avian cells (such as chicken embryo fibroblasts(CEF) prepared from chicken embryos obtained from fertilized eggs) orimmortalized avian cell lines, and in particular a Cairina moschata cellline immortalized with a duck TERT gene (see e.g. WO2010/130756 andWO2012/001075); avian cell line produced according to the processdescribed in WO2007/077256 or WO2009/004016; avian cell lineimmortalized with a combination of viral and/or cellular genes (see e.g.WO2005/042728); a spontaneously immortalized cell (e.g. the chicken DF1cell line disclosed in U.S. Pat. No. 5,879,924); or immortalized cellswhich derive from embryonic cells by progressive severance from growthfactors and feeder layer (e.g. Ebx chicken cell lines disclosed inWO2005/007840 and WO2008/129058).

For other vaccinia virus or other poxvirus strains, in addition to avianprimary cells (such as CEF) and avian cell lines, many other non-aviancell lines are available for production, including human cell lines suchas HeLa (ATCC-CRM-CCL-2™ or ATCC-CCL-2.2™), MRC-5, HEK-293; hamster celllines such as BHK-21 (ATCC CCL-10), and Vero cells. In a preferredembodiment, vaccinia virus other than MVA is amplified in HeLa cells(see e.g. WO2010/130753).

Producer cells are preferably cultivated in a medium free from animal-or human-derived products, using a chemically defined medium with noproduct of animal or human origin. In particular, while growth factorsmay be present, they are preferably recombinantly produced and notpurified from animal material. An appropriate animal-free medium may beeasily selected by those skilled in the art depending on selectedproducer cells. Such media are commercially available. In particular,when CEFs are used as producer cells, they may be cultivated in VP-SFMcell culture medium (Invitrogen). Producer cells are preferablycultivated at a temperature comprised between +30° C. and +38° C. (morepreferably at about +37° C.) for between 1 and 8 days (preferably for 1to 5 days for CEF and 2 to 7 days for immortalized cells) beforeinfection. If needed, several passages of 1 to 8 days may be made inorder to increase the total number of cells.

In step b), producer cells are infected by the viral vector underappropriate conditions (in particular using an appropriate multiplicityof infection (MOI) to permit productive infection of producer cells. Inparticular, when the therapeutic vaccine is based on MVA and isamplified using CEF, it may be seeded in the cell culture vesselcontaining CEFs at a MOI which is preferably comprised between 0.001 and0.1 (more preferably about 0.05). Infection step is also preferablyperformed in a medium (which may be the same as or different from themedium used for culture of producer cells) free from animal- orhuman-derived products, using a chemically defined medium with noproduct of animal or human origin.

In step c), infected producer cells are then cultured under appropriateconditions well known to those skilled in the art until progeny viralvector (e.g. infectious virus particles) is produced. Culture ofinfected producer cells is also preferably performed in a medium (whichmay be the same as or different from the medium used for culture ofproducer cells and/or for infection step) free from animal- orhuman-derived products (using a chemically defined medium with noproduct of animal or human origin) at a temperature between +30° C. and+37° C., for 1 to 5 days.

In step d), the viral vector produced in step c) is collected from theculture supernatant and/or the producer cells. Recovery from producercells (and optionally also from culture supernatant), may require a stepallowing the disruption of the producer cell membrane to allow theliberation of the vector from producer cells. The disruption of theproducer cell membrane can be induced by various techniques well knownto those skilled in the art, including but not limited to: freeze/thaw,hypotonic lysis, sonication, microfluidization, or high speedhomogenization.

Viral vectors may then be further purified, using purification stepswell known in the art. Various purification steps can be envisaged,including clarification, enzymatic treatment (e.g. endonuclease,protease, etc), chromatographic and filtration steps. Appropriatemethods are described in the art (e.g. WO2007/147528; WO2008/138533,WO2009/100521, WO2010/130753, WO2013/022764).

Immune Checkpoint Modulator(s)

Immune checkpoints and modulators thereof as well as methods of usingsuch compounds are described in the literature. “Immune checkpoint”proteins are directly or indirectly involved in an immune pathway thatunder normal physiological conditions is crucial for preventinguncontrolled immune reactions and thus for the maintenance ofself-tolerance and/or tissue protection. But under pathologicalconditions, they play a critical role in T cell exhaustion.

The one or more immune checkpoint modulator(s) in use herein mayindependently act at any step of the T cell-mediated immunity includingclonal selection of antigen-specific cells, T cell activation,proliferation, trafficking to sites of antigen and inflammation,execution of direct effector function and signaling through cytokinesand membrane ligands. Each of these steps is regulated bycounterbalancing stimulatory and inhibitory signals that in fine tunethe response. In the context of the present invention, the termencompasses (i) immune checkpoint modulator(s) capable ofdown-regulating at least partially the function of an inhibitory immunecheckpoint (e.g. by direct binding or inhibition of a ligand binding tosaid targeted immune checkpoint) so as to exert an antagonist functionand, thus antagonize an immune checkpoint-mediated inhibitory signal aswell as (ii) immune checkpoint modulator(s) capable of up-regulating atleast partially the function of a stimulatory immune checkpoint so as toexert an agonist function and, thus, amplify an immunecheckpoint-mediated stimulatory signal.

The one or more immune checkpoint modulator(s) in use herein mayindependently be a polypeptide or a nucleic acid molecule; with aspecific preference for peptide ligands, soluble domains of naturalreceptors, RNAi, antisense molecules, antibodies and protein scaffolds.

In a preferred embodiment, the immune checkpoint modulator is anantibody. In the context of the invention, “antibody” (“Ab”) is used inthe broadest sense and encompasses naturally occurring and engineered byman as well as full length antibodies or functional fragments or analogsthereof that are capable of binding the target immune checkpoint orepitope (thus retaining the target-binding portion). The antibody in usein the invention can be of any origin, e.g. human, humanized, animal(e.g. rodent or camelid antibody) or chimeric. It may be of any isotype(e.g. IgG1, IgG2, IgG3, IgG4, IgM, etc.). In addition, it may beglycosylated or non-glycosylated. The term antibody also includesbispecific or multispecific antibodies so long as they exhibit thebinding specificity described herein.

For illustrative purposes, full length antibodies are glycoproteinscomprising at least two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (VH) and a heavy chain constant region whichis made of three CH1, CH2 and CH3 domains (optionally with a hingebetween CH1 and CH2). Each light chain is comprised of a light chainvariable region (VL) and a light chain constant region which comprisesone CL domain. The VH and VL regions comprise hypervariable regions,named complementarity determining regions (CDR), and interspersed withmore conserved regions named framework regions (FR). Each VH and VL iscomposed of three CDRs and four FRs in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDR regions of the heavy and lightchains are determinant for the binding specificity.

As used herein, a “humanized antibody” refers to a non-human (e.g.murine, camel, rat, etc) antibody whose protein sequence has beenmodified to increase its similarity to a human antibody (i.e. producednaturally in humans). The process of humanization is well known in theart (see e.g. Pestra et al., 1997, Cancer Res. 57(20): 4593-9; U.S. Pat.No. 5,225,539; U.S. Pat. No. 5,530,101; U.S. Pat. No. 6,180,370;WO2012/110360). For example, a monoclonal antibody developed for humanuse can be humanized by substituting one or more residue of the FRregions to look like human immunoglobulin sequence whereas the vastmajority of the residues of the variable regions (especially the CDRs)are not modified and correspond to those of a non-human immunoglobulin.For general guidance, the number of these amino acid substitutions inthe FR regions is typically no more than 20 in each variable region VHor VL.

As used herein, a “chimeric antibody” refers to an antibody comprisingone or more element(s) of one species and one or more element(s) ofanother species, for example, a non-human antibody comprising at least aportion of a constant region (Fc) of a human immunoglobulin.

Antibody fragments can be engineered for use in the combination of theinvention. Representative examples include without limitation Fab, Fab′,F(ab′)2, dAb, Fd, Fv, scFv, di-scFv, diabody and any other artificialantibody. More specifically:

-   -   (i) a Fab fragment is represented by a monovalent fragment        consisting of the VL, VH, CL and CH1 domains;    -   (ii) a F(ab′)2 fragment is represented by a bivalent fragment        comprising two Fab fragments linked by at least one disulfide        bridge at the hinge region;    -   (iii) a Fd fragment consists of the VH and CH1 domains;    -   (iv) a Fv fragment consists of the VL and VH domains of a single        arm of an antibody,    -   (v) a dAb fragment consists of a single variable domain fragment        (VH or VL domain);    -   (vi) a single chain Fv (scFv) comprises the two domains of a Fv        fragment, VL and VH, that are fused together, optionally with a        linker to make a single protein chain (see e.g. Bird et al.,        1988, Science 242: 423-6; Huston et al., 1988, Proc. Natl. Acad.        Sci. USA 85: 5879-83; U.S. Pat. No. 4,946,778; U.S. Pat. No.        5,258,498); and    -   (vii) any other artificial antibody.

Methods for preparing antibodies, fragments and analogs thereof areknown in the art (see e.g. Harlow and Lane, 1988, Antibodies—Alaboratory manual; Cold Spring Harbor Laboratory, Cold Spring HarborN.Y.). In one embodiment, such an antibody can be generated a hostanimal with the targeted immune checkpoint modulator. Alternatively, itcan be produced from hybridomas (see e.g. Kohler and Milstein, 1975,Nature 256: 495-7; Cote et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-30; Cole et al. in Monoclonal antibodies and Cancer Therapy; AlanLiss pp77-96), recombinant techniques (e.g. using phage displaymethods), peptide synthesis and enzymatic cleavage. Antibody fragmentscan be produced by recombinant technique as described herein. They mayalso be produced by proteolytic cleavage with enzymes such as papain toproduce Fab fragments or pepsin to produce F(ab′)2 fragments asdescribed in the literature (see e.g. Wahl et al., 1983, J. Nucl. Med.24: 316-25). Analogs (or fragment thereof) can be generated byconventional molecular biology methods (PCR, mutagenesis techniques). Ifneeded, such fragments and analogs may be screened for functionality inthe same manner as intact antibodies (e.g. by standard ELISA assay).

In a preferred embodiment, at least one of the one or more immunecheckpoint modulator(s) for use in the present invention is a monoclonalantibody, with a specific preference for a human (in which both theframework regions are derived from human germline immunoglobinsequences) or a humanized antibody according to well-known humanizationprocess.

Desirably, the one or more immune checkpoint modulator(s) in use in thepresent invention antagonizes at least partially (e.g. more than 50%)the activity of inhibitory immune checkpoint(s), in particular thosemediated by any of the following PD-1, PD-L1, PD-L2, LAG3, Tim3, BTLA,SLAM, 2B4, CD160, KLRG-1 and CTLA4, with a specific preference for ahuman or humanized monoclonal antibody that specifically binds to any ofsuch target proteins. The term “specifically binds to” refers to thecapacity to a binding specificity and affinity for a particular targetor epitope even in the presence of a heterogeneous population of otherproteins and biologics. Thus, under designated assay conditions, theantibody in use in the invention binds preferentially to its target anddoes not bind in a significant amount to other components present in atest sample or subject. Preferably, such an antibody shows high affinitybinding to its target with an equilibrium dissociation constant equal orbelow 1×10⁻⁶M (e.g. at least 0.5×10⁻⁶, 1×10⁻⁷, 1×10⁻⁸, 1×10⁻⁹, 1×10⁻¹⁰,etc). Alternatively, the one or more immune checkpoint modulator(s) inuse in the present invention exerts an agonist function in the sensethat it is capable of stimulating or reinforcing stimulatory signals, inparticular those mediated by CD28 with a specific preference for any ofICOS, CD137 (4-1BB), OX40, CD27, CD40 and GITR immune checkpoints.Standard assays to evaluate the binding ability of the antibodies towardimmune checkpoints are known in the art, including for example, ELISAs,Western blots, RIAs and flow cytometry. The binding kinetics (e.g.,binding affinity) of the antibodies also can be assessed by standardassays known in the art, such as by Biacore analysis.

In a preferred embodiment, at least one of the one or more checkpointmodulator(s) for use in this invention is an antibody capable ofantagonizing at least partially the protein Programmed Death 1 (PD-1),and especially an antibody that specifically binds to human PD-1. PD-1is part of the immunoglobulin (Ig) gene superfamily and a member of theCD28 family. It is a 55 kDa type 1 transmembrane protein expressed onantigen-experienced cells (e.g. activated B cells, T cells, and myeloidcells) (Agata et al., 1996, Int. Immunol. 8: 765-72; Okazaki et al.,2002, Curr. Opin. Immunol. 14: 391779-82; Bennett et al., 2003, J.Immunol 170: 711-8). In normal context, it acts by limiting the activityof T cells at the time of inflammatory response, thereby protectingnormal tissues from destruction (Topalian, 2012, Curr. Opin. Immunol.24: 207-12). Two ligands have been identified for PD-1, respectivelyPD-L1 (programmed death ligand 1) and PD-L2 (programmed death ligand 2)(Freeman et al., 2000, J. Exp. Med. 192: 1027-34; Carter et al., 2002,Eur. J. Immunol. 32: 634-43). PD-L1 was identified in 20-50% of humancancers (Dong et al., 2002, Nat. Med. 8: 787-9). The interaction betweenPD-1 and PD-L1 resulted in a decrease in tumor infiltrating lymphocytes,a decrease in T-cell receptor mediated proliferation, and immune evasionby the cancerous cells (Dong et al., 2003, J. Mol. Med. 81: 281-7; Blanket al., 2005, Cancer Immunol. Immunother. 54: 307-314). The completenucleotide and amino acid PD-1 sequences can be found under GenBankAccession No U64863 and NP_005009.2. A number of anti PD1 antibodies areavailable in the art (see e.g. those described in WO2004/004771;WO2004/056875; WO2006/121168; WO2008/156712; WO2009/014708;WO2009/114335; WO2013/043569; and WO2014/047350). Preferred anti PD-1antibodies in the context of this invention are FDA approved or underadvanced clinical development and one may use in particular an anti-PD-1antibody selected from the group consisting of Nivolumab (also termedBMS-936558 under development by Bristol Myer Squibb), Lanbrolizumab(also termed MK-3475 under development by Merck), and Pidilizumab (alsotermed CT-011 under development by CureTech).

Another preferred example of immune checkpoint modulator is representedby a modulator capable of antagonizing at least partially the PD-1ligand termed PD-L1, and especially an antibody that recognizes humanPD-L1. A number of anti PD-L1 antibodies are available in the art (seee.g. those described in EP1907000). Preferred anti PD-L1 antibodies areFDA approved or under advanced clinical development (e.g. MPDL3280Aunder development by Genentech/Roche and BMS-936559 under development byBristol Myer Squibb).

Still another preferred example of immune checkpoint modulator isrepresented by a modulator capable of antagonizing at least partiallythe CTLA-4 protein, and especially an antibody that recognizes humanCTLA-4. CTLA4 (for cytotoxic T-lymphocyte-associated antigen 4) alsoknown as CD152 was identified in 1987 (Brunet et al., 1987, Nature 328:267-70) and is encoded by the CTLA4 gene (Dariavach et al., Eur. J.Immunol. 18: 1901-5). It is expressed on the surface of T cells where itprimarily regulates the amplitude of the early stages of T cellactivation. Recent work has suggested that CTLA-4 may function in vivoby capturing and removing B7-1 and B7-2 from the membranes ofantigen-presenting cells, thus making these unavailable for triggeringof CD28 (Qureshi et al., Science, 2011, 332: 600-3). The complete CTLA-4nucleic acid sequence can be found under GenBank Accession No LI 5006. Anumber of anti CTLA-4 antibodies are available in the art (see e.g.those described in U.S. Pat. No. 8,491,895). Preferred anti CTLA-4antibodies in the context of this invention are FDA approved or underadvanced clinical development. One may cite more particularly ipilimumabmarketed by Bristol Myer Squibb as Yervoy (see e.g. U.S. Pat. No.6,984,720; U.S. Pat. No. 8,017,114), tremelimumab under development byPfizer (see e.g. U.S. Pat. No. 7,109,003 and U.S. Pat. No. 8,143,379)and single chain anti-CTLA4 antibodies (see e.g. WO97/20574 andWO2007/123737).

Immune checkpoint modulator for antagonizing the TIM3 receptor may alsobe used in the combination product of the present invention (see e.g.Ngiow et al., 2011, Cancer Res. 71: 3540-51; US2012-0189617).

Another Immune checkpoint modulator for antagonizing the LAG3 receptormay also be used in the combination of the present invention (see e.g.Toni-Jun et al., 2011, 2014, ACCR poster LB266; Woo et al., 2012, CancerRes. 72: 917-27).

Still another example of immune checkpoint modulator is represented byan OX40 agonist such as agonist ligand of OX40 (OX40L) (see e.g. U.S.Pat. No. 5,457,035, U.S. Pat. No. 7,622,444; WO03/082919) or an antibodydirected to the OX40 receptor (see e.g. U.S. Pat. No. 7,291,331 andWO03/106498).

Other examples of immune checkpoint modulators are represented byanti-KIR or anti-CD96 antibody targeting the inhibitory receptorsharboured by CD8+ T cells and NK cells.

The present invention encompasses a combination comprising more than oneimmune checkpoint modulator(s). A preferred example includes withoutlimitation using an anti-CTLA-4 antibody with an anti-PD-1 or ananti-PD-L1 antibody in combination with the therapeutic vaccinedescribed herein.

Nucleic acid molecules encoding the relevant portion(s) of the desiredimmune checkpoint modulator can be obtained by standard molecularbiology techniques using sequence data accessible in the art and theinformation provided herein. For example, cDNAs encoding the light andheavy chains of the antibody or their CDRs can be isolated from theproducing hybridoma, immunoglobulin gene libraries or any availablesource.

In one embodiment, the one or more immune checkpoint modulator(s) foruse in this invention can be comprised in the therapeutic vaccinedescribed herein. For example, the encoding nucleic acid molecule can beinserted in a vector-based therapeutic vaccine (e.g. in anantigen-encoding viral vector). In this context, the nucleic acidmolecules encoding the polypeptide(s) of interest and the immunecheckpoint modulator(s) are preferably expressed independently usingdistinct regulatory elements. Alternatively, the one or more immunecheckpoint modulator(s) for use in this invention can be expressed froman independent vector system such as one of those described herein inconnection with the therapeutic vaccine for separate or concomitantadministration to the subject in need thereof.

Still alternatively, the one or more immune checkpoint modulator(s) foruse in this invention can be produced by recombinant means usingsuitable expression vectors and host cells for administration asrecombinant polypeptide to the subject in need thereof.

Production of Immune Checkpoint Modulator

Insertion into the expression vector can be performed by routinemolecular biology, e.g. as described in Sambrook et al. (2001, MolecularCloning-A Laboratory Manual, Cold Spring Harbor Laboratory). Insertioninto a virus-based therapeutic vaccine can be performed throughhomologous recombination as described in Chartier et al. (1996, J.Virol. 70: 4805-10) and Paul et al. (2002, Cancer Gene Ther. 9: 470-7).

A variety of host-vector systems may be used or constructed to expressthe one or more immune checkpoint modulator(s) for use in the presentinvention, including prokaryotic organisms such as bacteria (e.g. E.coli or Bacillus subtilis); yeast (e.g. Saccharomyces cerevisiae,Saccharomyces pombe, Pichia pastoris); insect cell systems (e.g. Sf 9cells and baculovirus); plant cell systems (e.g. cauliflower mosaicvirus CaMV; tobacco mosaic virus TMV) and mammalian cell systems (e.g.cultured cells). Typically, such vectors are commercially available(e.g. in Invitrogen, Stratagene, Amersham Biosciences, Promega, etc.) oravailable from depositary institutions such as the American Type CultureCollection (ATCC, Rockville, Md.) or have been the subject of numerouspublications describing their sequence, organization and methods ofproducing, allowing the artisan to apply them. For general purposes,such vectors usually comprise one or more element(s) enablingmaintenance, propagation or expression of the nucleic acid molecule inthe host cell. Representative elements include without limitation markergene(s) in order to facilitate identification and isolation of therecombinant host cells (e.g. by complementation of a cell auxotrophy orby antibiotic resistance), stabilizing elements (e.g. DAP system asdescribed in U.S. Pat. No. 5,198,343), and integrative elements (e.g.LTR viral sequences and transposons).

Suitable plasmid vectors for use in prokaryotic systems include withoutlimitation pBR322 (Gibco BRL), pUC (Gibco BRL), pbluescript(Stratagene), p Poly (Lathe et al., 1987, Gene 57: 193-201), pTrc (Amannet al., 1988, Gene 69: 301-15); pET lid (Studier et al., 1990, GeneExpression Technology: Methods in Enzymology 185: 60-89); pIN (Inouye etal., 1985, Nucleic Acids Res. 13: 3101-9; Van Heeke et al., 1989, J.Biol. Chem. 264: 5503-9); and pGEX vectors where the nucleic acidmolecule can be expressed in fusion with glutathione S-transferase (GST)(Amersham Biosciences Product). Suitable vectors for expression in yeast(e.g. S. cerevisiae) include, but are not limited to pYepSec1 (Baldariet al., 1987, EMBO J. 6: 229-34), pMFa (Kujan et al., 1982, Cell 30:933-43), pJRY88 (Schultz et al., 1987, Gene 54: 113-23), pYES2(Invitrogen Corporation) and pTEF-MF (Dualsystems Biotech Product).Plasmid and viral vectors such as those described herein in connectionwith the therapeutic vaccine may also be used to produce the immunecheckpoint modulator(s) by recombinant means.

Recombinant DNA technologies can also be used to improve expression ofthe nucleic acid molecule encoding the immune checkpoint modulator inthe host cell, e.g. by using high-copy number vectors, substituting ormodifying one or more transcriptional regulatory sequences (e.g.promoter, enhancer and the like), optimizing the codon usage andsuppressing negative sequences that may destabilize the transcript asdescribed herein in connection with the nucleic acid molecule(s)encoding the polypeptide(s) of interest).

As before, the nucleic acid molecule encoding the immune checkpointmodulator is in a form suitable for its expression in a host cell, whichmeans that the nucleic acid molecule is placed under the control of oneor more regulatory sequences, appropriate to the vector, the host celland/or the level of expression desired as described above. Constitutivepromoters (e.g. PGK, CMV promoters, etc), inducible eukaryotic promotersregulated by exogenously supplied compounds (e.g. TRP and IPTG-induciblepTAC promoters, zinc-inducible metallothionein (MT) promoter,dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter,tetracycline-repressible and rapamycin-inducible promoter, etc) can beused as well as any of the promoters described hereinafter forexpression of nucleic acid molecule encoding the polypeptide ofinterest.

The methods for the recombinant production of the immune checkpointmodulator are conventional in the art. Typically such methods comprise(a) introducing the expression vector described herein into a suitableproducer cell to produce a transfected or infected producer cell, (b)culturing in-vitro said transfected or infected producer cell underconditions suitable for its growth, (c) recovering the immune checkpointmodulator from the cell culture, and (d) optionally, purifying therecovered immune checkpoint modulator.

In the context of the invention, producer cells include prokaryoticcells, lower eukaryotic cells such as yeast, and other eukaryotic cellssuch as insect cells, plant and mammalian (e.g. human or non-human)cells. Preferred E. coli cells include without limitation E. coli BL21(Amersham Biosciences). Preferred yeast producer cells include withoutlimitation S. cerevisiae, S. pombe, Pichia pastoris. Preferred mammalianproducer cells include without limitation BHK-21 (baby hamster kidney),CV-1 (African monkey kidney cell line), COS (e.g. COS-7) cells, Chinesehamster ovary (CHO) cells, mouse NIH/3T3 cells, HeLa cells, Vero cells,HEK293 cells and PERC.6 cells as well as the corresponding hybridomacells.

Transfection/infection of producer host cells is conventional and mayuse additional compounds so as to improve the transfection efficiencyand/or stability of the vector. These compounds are widely documented inthe literature such as polycationic polymers (e.g. chitosan,polymethacrylate, PEI, etc), cationic lipids (e.g. DC-Chol/DOPE,transfectam lipofectin now available from Promega) and liposomes.

The producer cells can be cultured in conventional fermentationbioreactors, flasks, and petri plates. Culturing can be carried out at atemperature, pH and oxygen content appropriate for a given host cell. Noattempts to describe in detail the various methods known for theproduction of proteins in prokaryote and eukaryote cells will be madehere. Production of the immune checkpoint modulator can be periplasmic,intracellular or preferably secreted outside the producer cell (e.g. inthe culture medium). If necessary, especially when the immune checkpointmodulator is not secreted outside the producer cell or where it is notsecreted completely, it can be recovered by standard lysis procedures,including freeze thaw, sonication, mechanical disruption, use of lysingagents and the like. If secreted, it can be recovered directly from theculture medium.

Optionally, the immune checkpoint modulator can then be purified bywell-known purification methods including ammonium sulfateprecipitation, acid extraction, gel electrophoresis, filtration andchromatographic methods (e.g. reverse phase, size exclusion, ionexchange, affinity, phosphocellulose, hydrophobic-interaction orhydroxylapatite chromatography, etc). The conditions and technology usedto purify a particular protein will depend on factors such as netcharge, molecular weight, hydrophobicity, hydrophilicity and will beapparent to those having skill in the art. Moreover, the level ofpurification will depend on the intended use. It is also understood thatdepending upon the producer cell, the immune checkpoint modulatorproteins can have various glycosylation patterns, or may benon-glycosylated (e.g. when produced in bacteria) as described herein.

Desirably, the immune checkpoint modulator in use in the presentinvention is at least partially purified in the sense that it issubstantially free of other antibodies having different antigenicspecificities and/or other cellular material. Further, the immunecheckpoint modulator may be formulated according to the conditionsconventionally used in the art (e.g. WO2009/073569).

In accordance with the present invention, a variety of modifications canbe introduced in the immune checkpoint inhibitor so as to increase itsbiological half-life, its affinity, its stability and/or its production.For example, a signal peptide may be included for facilitating secretionof the immune checkpoint modulator in a cell culture as describedherein. As an additional example, a tag peptide (typically a shortpeptide sequence able to be recognized by available antisera orcompounds) may also be added for facilitating purification of therecombinant immune checkpoint modulator. A vast variety of tag peptidescan be used in the context of the invention including, withoutlimitation, PK tag, FLAG octapeptide, MYC tag, HIS tag (usually astretch of 4 to 10 histidine residues) and e-tag (U.S. Pat. No.6,686,152). The tag peptide(s) may be independently positioned at theN-terminus of the protein or alternatively at its C-terminus oralternatively internally or at any of these positions when several tagsare employed. Tag peptides can be detected by immunodetection assaysusing anti-tag antibodies.

Another approach that may be pursued in the context of the presentinvention is coupling of the immune checkpoint modulator to an externalagent such as a radiosensitizer agent, a cytotoxic agent and/or alabelling agent. The coupling can be covalent or not. As used herein,the term “radiosensitizer” refers to a molecule that makes cells moresensitive to radiation therapy. Radiosensitizer includes, but are notlimited to, metronidazole, misonidazole, desmethylmisonidazole,pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233,E09, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR),5-iododeoxyuhdine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FUdR),hydroxyurea and cisplatin. As used herein, the term “cytotoxic agent”refers to a compound that is directly toxic to cells, preventing theirreproduction or growth such as toxins (e.g. an enzymatically activetoxin of bacterial, fungal, plant or animal origin, or fragmentsthereof). As used herein, “a labeling agent” refers to a detectablecompound. The labeling agent may be detectable by itself (e.g.,radioactive isotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical modification of a substratecompound which is detectable.

Another modification is pegylation for example to increase thebiological half-life of the antibody. Methods for pegylating proteinsare known in the art (see e.g. EP154316; EP401384; WO98/15293,WO01/23001, etc).

Combination Product and Therapy

The term “combination therapy” and any variation such as “combined use”refers to the action of administering in the same subject at least thetwo entities being an object of the invention and described herein.

In one embodiment, the present invention relates to a combinationproduct in the form of a composition comprising a therapeuticallyeffective amount of at least the therapeutic vaccine and one or moreimmune checkpoint modulator entities described herein and apharmaceutically acceptable vehicle. In another embodiment, the presentinvention relates to distinct compositions for combined use, onecomprising at least a therapeutically effective amount of thetherapeutic vaccine and a pharmaceutically acceptable vehicle andanother comprising a therapeutically effective amount of the one or moreimmune checkpoint modulator and a pharmaceutically acceptable vehicle.One may proceed with one or more administration(s) of each entity (orcomposition(s) thereof) which can be concomitant, sequential orinterspersed via the same or different routes.

A “therapeutically effective amount” corresponds to the amount of eachof the active entities (therapeutic vaccine and the one or more immunecheck point modulator(s)) comprised in the combination or composition(s)of the invention that is sufficient for producing one or more beneficialresults. Such a therapeutically effective amount may vary as a functionof various parameters such as the mode of administration; the age andweight of the subject; the nature and extent of symptoms; the ability ofthe subject to respond to the treatment, kind of concurrent treatment;the frequency of treatment and/or the need for prevention or therapy,etc.

When “prophylactic” use is concerned, the combination is administered ata dose sufficient to prevent or to delay the onset and/or establishmentand/or relapse of a pathologic condition, especially in a subject atrisk. For “therapeutic” use, the therapeutic vaccine and the immunecheckpoint modulator(s) are both administered to a subject diagnosed ashaving a disease or pathological condition with the goal of treating it,optionally in association with one or more conventional therapeuticmodalities.

The term “pharmaceutically acceptable vehicle” is intended to includeany and all carriers, solvents, diluents, excipients, adjuvants,dispersion media, coatings, antibacterial and antifungal agents,absorption agents and the like compatible with administration in mammalsand in particular human subjects.

Each of the therapeutic vaccine and the one or more immune check pointmodulator(s) or composition(s) thereof can independently be placed in asolvent or diluent appropriate for human or animal use. In particular,each or both may be formulated so as to ensure its stability inparticular under the conditions of manufacture and long-term storage(i.e. for at least 6 months, with a preference for at least two years)at freezing (e.g. −70° C., −20° C.), refrigerated (e.g. 4° C.) orambient (e.g. 20-25° C.) temperature. Such formulations generallyinclude a liquid carrier such as aqueous solutions. Physiological salinesolution, Ringer's solution, Hank's solution, saccharide solution (e.g.glucose, trehalose, saccharose, dextrose, etc) and other aqueousphysiologically balanced salt solutions may be used (see for example themost current edition of Remington: The Science and Practice of Pharmacy,A. Gennaro, Lippincott, Williams&Wilkins). Animal or vegetable oils,mineral or synthetic oils are also suitable. Advantageously, theformulation appropriate for the therapeutic vaccine and the one or moreimmune check point modulator(s) or composition(s) thereof is suitablybuffered for human use, preferably at physiological or slightly basic pH(e.g. from approximately pH 7 to approximately pH 9 with a specificpreference for a pH comprised between 7 and 8 and more particularlyclose to 7.5). Suitable buffers include without limitation TRIS(tris(hydroxymethyl)methylamine), TRIS-HCl(tris(hydroxymethyl)methylamine-HCl), HEPES(4-2-hydroxyethyl-1-piperazineethanesulfonic acid), phosphate buffer(e.g. PBS), ACES (N-(2-Acetamido)-aminoethanesulfonic acid), PIPES(Piperazine-N,N′-bis(2-ethanesulfonic acid)), MOPSO(3-(N-Morpholino)-2-hydroxypropanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), TES(2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), DIPSO(3-[bis(2-hydroxyethyl)amino]-2-hydroxypropane-1-sulfonic acid), MOBS(4-(N-morpholino)butanesulfonic acid), TAPSO(3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic Acid),HEPPSO (4-(2-Hydroxyethyl)-piperazine-1-(2-hydroxy)-propanesulfonicacid), POPSO(2-hydroxy-3-[4-(2-hydroxy-3-sulfopropyl)piperazin-1-yl]propane-1-sulfonicacid), TEA (triethanolamine), EPPS(N-(2-Hydroxyethyl)-piperazine-N′-3-propanesulfonic acid), and TRICINE(N-[Tris(hydroxymethyl)-methyl]-glycine). Preferably, said buffer isselected from TRIS-HCl, TRIS, Tricine, HEPES and phosphate buffercomprising a mixture of Na₂HPO₄ and KH₂PO₄ or a mixture of Na₂HPO₄ andNaH₂PO₄. Said buffer (in particular those mentioned above and notablyTRIS-HCl) is preferably present in a concentration of 10 to 50 mM. Itmight be beneficial to also include in such formulations a monovalentsalt so as to ensure an appropriate osmotic pressure. Said monovalentsalt may notably be selected from NaCl and KCl, preferably saidmonovalent salt is NaCl, preferably in a concentration of 10 to 500 mM.

The formulation appropriate for use in the context of the presentinvention, and especially liquid or frozen formulation, may also includea cryoprotectant so as to protect the therapeutic vaccine and/or the oneor more immune check point modulator(s) (in particular virus-basedcomposition) at low storage temperature, such as at about +5° C. andlower. Suitable cryoprotectants include without limitation sucrose (orsaccharose), trehalose, maltose, lactose, mannitol, sorbitol andglycerol, preferably in a concentration of 0.5 to 20% (weight ing/volume in L, referred to as w/v). For example, sucrose is preferablypresent in a concentration of 5 to 15% (w/v), with a specific preferencefor about 10%.

The formulation appropriate for use in the present invention andespecially liquid formulation may further comprise a pharmaceuticallyacceptable chelating agent, and in particular an agent chelatingdications for improving stability. The pharmaceutically acceptablechelating agent may notably be selected from ethylenediaminetetraaceticacid (EDTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid(BAPTA), ethylene glycol tetraacetic acid (EGTA), dimercaptosuccinicacid (DMSA), diethylene triamine pentaacetic acid (DTPA), and2,3-Dimercapto-1-propanesulfonic acid (DMPS). The pharmaceuticallyacceptable chelating agent is preferably present in a concentration ofat least 50 μM with a specific preference for a concentration of 50 to1000 μM. Preferably, said pharmaceutically acceptable chelating agent isEDTA present in a concentration close to 150 μM.

Additional compounds may further be present to increase stability of theformulated therapeutic vaccine and/or immune check point modulator(s) orcomposition(s) thereof. Such additional compounds include, withoutlimitation, C₂-C₃ alcohol (desirably in a concentration of 0.05 to 5%(volume/volume or v/v)), sodium glutamate (desirably in a concentrationlower than 10 mM), non-ionic surfactant (Evans et al. 2004, J Pharm Sci.93:2458-75, Shi et al., 2005, J Pharm Sci. 94:1538-51, U.S. Pat. No.7,456,009, US2007/0161085) such as Tween 80 (also known as polysorbate80) at low concentration below 0.1%. Divalent salts such as MgCl₂ orCaCl₂ have been found to induce stabilization of various biologicalproducts in the liquid state (see Evans et al. 2004, J Pharm Sci.93:2458-75 and U.S. Pat. No. 7,456,009). Amino acids, and in particularhistidine, arginine or methionine, have been found to inducestabilization of various viruses in the liquid state (see Evans et al.,2004, J Pharm Sci. 93:2458-75, U.S. Pat. No. 7,456,009, US2007/0161085,U.S. Pat. No. 7,914,979, WO2014/029702 and WO2014/053571).

The presence of high molecular weight polymers such as dextran orpolyvinylpyrrolidone (PVP) is particularly suited for freeze-driedformulations. Lyophilized formulations are generally obtained by aprocess involving vacuum drying and freeze-drying (see e.g. WO03/053463;WO2006/0850082; WO2007/056847; WO2008/114021) and the presence of thesepolymers assists in the formation of the cake during freeze-drying (seeEP1418942 and WO2014/053571).

Various formulations available in the art either in frozen, liquid orfreeze-dried form can be independently used to preserve the therapeuticvaccine and/or immune check point modulator(s) or composition(s) thereof(e.g. WO98/02522, WO00/29024, WO00/34444, WO01/66137, WO03/053463,WO2006/0850082, WO2007/056847 and WO2008/114021, etc). For illustrativepurposes, sterile histidine, acetate citrate or phosphate buffers salinecontaining surfactant such as polysorbate 80 and protectants such assucrose or mannitol are adapted to the preservation of recombinantantibodies and buffered formulations including NaCl and/or sugar areparticularly adapted to the preservation of vectorised therapeuticvaccine (e.g. Tris-HCl 10 mM pH 8 with saccharose 5% (w/v), Sodiumglutamate 10 mM, and NaCl 50 mM or phosphate-buffered saline withglycerol (10%) and NaCl).

Formulation can be adapted according to the mode of administration toensure proper distribution or delayed release in vivo. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylacticacid and polyethylene glycol. Many methods for the preparation of suchformulations are described in the art (e.g. J. R. Robinson in “Sustainedand Controlled Release Drug Delivery Systems”, ed., Marcel Dekker, Inc.,New York, 1978; WO01/23001; WO2006/93924; WO2009/53937).Gastro-resistant capsules and granules are particularly appropriate fororal administration, suppositories for rectal or vaginal administration,optionally in combination with absorption enhancers useful to increasethe pore size of the mucosal membranes. Such absorption enhancers aretypically substances having structural similarities to the phospholipiddomains of the mucosal membranes (such as sodium deoxycholate, sodiumglycocholate, dimethyl-beta-cyclodextrin,lauryl-1-lysophosphatidylcholine).

Each of the therapeutic vaccine and/or the immune check pointmodulator(s) or composition(s) thereof may also contain pharmaceuticallyacceptable excipients for providing desirable pharmaceutical orpharmacodynamic properties, including for example osmolarity, viscosity,clarity, colour, sterility, stability, dissolution, release orabsorption into the subject, or delivery to a particular organ.

The appropriate dosage of the therapeutic vaccine and the immunecheckpoint modulator(s) as well as the optimal ratios of each entity maybe determined by techniques well known in the art. Further refinement ofthe calculations necessary to adapt the appropriate dosage for a subjector a group of subjects may be routinely made by a practitioner, in thelight of the relevant circumstances.

Suitable dosage of the immune checkpoint modulator(s) varies from about0.01 mg/kg to about 50 mg/kg, advantageously from about 0.1 mg/kg toabout 30 mg/kg, desirably from about 0.5 mg/kg to about 25 mg/kg,preferably from about 1 mg/kg to about 20 mg/kg, more preferably fromabout 2 mg/kg to about 15 mg/kg, with a specific preference for dosesfrom about 3 mg/kg to about 10 mg/kg when used by parenteral injection.However, doses may be adapted to the administration route and thesubject to be treated by a factor of variation comprised between 1.5 and100. In some embodiments, two or more monoclonal antibodies withdifferent binding specificities are administered simultaneously, inwhich case the dosage of each antibody administered falls within theranges indicated.

Suitable dosage for a virus-based therapeutic vaccine varies fromapproximately 10⁵ to approximately 10¹³ vp (viral particles), iu(infectious unit) or pfu (plaque-forming units) depending on thequantitative technique used. As a general guidance, adenovirus dosesfrom approximately 10⁶ to approximately 5×10¹² vp are suitable,preferably from approximately 10⁷ vp to approximately 10¹² vp, morepreferably from approximately 10⁸ vp to approximately 5×10¹¹ vp; dosesof approximately 5×10⁸ vp to approximately 10¹¹ vp being particularlypreferred especially for human use. Individual doses which are suitablefor MVA-based therapeutic vaccine comprise from approximately 10⁴ toapproximately 10¹² pfu, preferably from approximately 10⁵ pfu toapproximately 10¹¹ pfu, more preferably from approximately 10⁶ pfu toapproximately 10¹⁰ pfu; doses of approximately 10⁷ pfu to approximately10⁸ pfu being particularly preferred especially for human use.Individual doses which are suitable for Vaccinia-based therapeuticvaccine comprise from approximately 10⁵ to approximately 10¹³ pfu,preferably from approximately 10⁶ pfu to approximately 10¹¹ pfu, morepreferably from approximately 10⁷ pfu to approximately 10¹⁰ pfu; dosesof approximately 10⁸ pfu to approximately 5×10⁹ vp being particularlypreferred especially for human use. The quantity of virus present in asample can be determined by routine titration techniques, e.g. bycounting the number of plaques following infection of permissive cells(e.g. 293 or PER6C6 for Ad, BHK-21 or CEF for MVA, HeLa for VV), bymeasuring the A260 absorbance (vp titers), or still by quantitativeimmunofluorescence, e.g. using anti-virus antibodies (iu titers).Suitable dosage for a plasmid-based therapeutic vaccine varies from 10μg to 20 mg, advantageously from 100 μg to 10 mg and preferably fromapproximately 0.5 mg to approximately 5 mg.

Administration

The combination product of the invention is suitable for singleadministration or a series of administrations. In particular whendistinct compositions are contemplated, the therapeutic vaccine and theimmune check point modulator(s) may be administered together orseparately to the subject and in a single dose or multiple doses.Administrations may be concomitant (e.g. mixed in the same compositionor in different compositions administered at approximately the sametime), sequential (therapeutic vaccine followed by immune checkpointmodulator or vice versa) or interspersed (intermixed administrations atvarious intervals) and performed by the same or different routes at thesame site or at alternative sites.

Any of the conventional administration routes is applicable in thecontext of the invention including parenteral, topical or mucosalroutes, for the combination product or composition(s) of the invention.Parenteral routes are intended for administration as an injection orinfusion and encompass systemic as well as local routes. Parenteralinjection types that may be used to administer the combination productof the invention are intravenous (into a vein, such as the portal veinfeeding liver), intravascular (into a blood vessel), intra-arterial(into an artery such as hepatic artery), intradermal (into the dermis),subcutaneous (under the skin), intramuscular (into muscle),intraperitoneal (into the peritoneum) and intratumoral (into a tumor orits close vicinity) or still by scarification. Infusions typically aregiven by intravenous route. Mucosal administrations include withoutlimitation oral/alimentary, intranasal, intratracheal, intrapulmonary,intravaginal or intra-rectal route. Topical administration can also beperformed using transdermal means (e.g. patch and the like). Preferredroutes of administration for the immune checkpoint modulator(s) includeintravenous (e.g. intravenous injection or infusion), and intratumoral.Preferred routes of administration for the therapeutic vaccine includeintravenous, intramuscular, subcutaneous and intratumoral. For example,intratumoral inoculations of the therapeutic vaccine could beadvantageously combined with intravenous injections of the immunecheckpoint modulator(s).

Administrations may use conventional syringes and needles (e.g.Quadrafuse injection needles) or any compound or device available in theart capable of facilitating or improving delivery of the active agent(s)in the subject (e.g. electroporation for facilitating intramuscularadministration). An alternative is the use of a needleless injectiondevice to administer at least one of the active entities comprised inthe combination product of the invention (e.g. Biojector™ device).Transdermal patches may also be envisaged.

In one embodiment, the therapeutic vaccine and the one or more immunecheckpoint modulator(s) or composition(s) thereof are administeredsequentially, such as the vaccines being administered first and theimmune checkpoint modulator(s) second, or vise-versa (immune checkpointmodulator(s) being administered first and the therapeutic vaccinesecond). The sequence may vary. For example, the order of theadministrations can be reversed or kept in the same order at each timepoint of administration.

One may also proceed by interspersed administrations of the therapeuticvaccine and the immune checkpoint modulator(s). The period of timebetween the first administration of the therapeutic vaccine and thefirst administration of the immune check point modulator(s) may varyfrom approximately several minutes to several week(s). It is alsopossible to proceed for each entity via sequential cycles ofadministrations that are repeated after a rest period. Intervals betweeneach administration can be from one hour to one year (e.g. 24 h, 48 h,72 h, weekly, every two weeks, monthly or yearly). Intervals can also beirregular (e.g. following the measurement of monoclonal antibodies inthe patient blood levels). The doses can vary for each administrationwithin the range described above. Preferably, the time interval betweeneach therapeutic vaccine administration can vary from approximately 1day to approximately 8 weeks, advantageously from approximately 2 daysto approximately 6 weeks, preferably from approximately 3 days toapproximately 4 weeks and even more preferably from approximately 1 weekto approximately 3 weeks with a specific preference for about one week.In combination, the time interval between each administration of immunecheck point modulator(s) can vary from approximately 2 days toapproximately 8 weeks, advantageously from approximately 1 week toapproximately 6 weeks, preferably every 3 weeks.

A preferred therapeutic scheme involves from 4 to 15 (4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14 or 15) administrations of 10⁷ or 10⁹ pfu of aMVA-based therapeutic vaccine at approximately 1 to 3 week intervalinterspersed with 2 to 6 administrations of 3 to 10 mg/kg of anti-immunecheckpoint antibody(ies)(s) every 2 or 3 weeks. For illustrativepurposes, a preferred administration schedule comprises subcutaneousadministrations of a MUC1-expressing MVA vector (such as TG4010) at adose of approximately 10⁸ pfu weekly for 6 weeks and then every threeweeks interspersed with intravenous administrations of an anti-CTLA4antibody such as ipilimumab at a dose of approximately 3 mg/kg every 3weeks for a total of four doses.

The combination product or composition of the invention is for use fortreating or preventing diseases or pathologic condition, especiallythose caused by a pathogenic organism or an unwanted cell divisionaccording to the modalities described herein. A “disease” (and any formof disease such as “disorder” or “pathological condition”) is typicallycharacterized by identifiable symptoms. Exemplary diseases include, butare not limited to, infectious diseases that result from an infectionwith a pathogenic organism (e.g. bacteria, parasite, virus, fungus, etc)and proliferative diseases involving abnormal proliferation of cellssuch as neoplastic diseases (e.g. cancer), rheumatoid arthritis andrestenosis.

The present invention also relates to a method of treatment comprisingadministering a combination product or composition(s) of therapeuticvaccine and/or one or more immune checkpoint modulator(s) in an amountsufficient to treat or prevent a disease or a pathologic condition in asubject in need thereof or alleviate one or more symptoms related to orassociated with the diseases and pathologic condition, according to themodalities described herein. In a preferred embodiment, the disease orpathologic condition to be treated is a proliferative or an infectiousdisease (e.g. especially a chronic infection). Accordingly, the presentinvention also relates to a method for inhibiting tumor cell growthcomprising administering a combination product or composition(s) oftherapeutic vaccine and/or one or more immune checkpoint modulator(s) toa subject in need thereof. In the context of the invention, the methodsand use according to the invention aim at slowing down, curing,ameliorating or controlling the occurrence or the progression of thetargeted disease.

As used herein, the term “cancer” includes, but is not limited to, solidtumors and blood borne tumors. The term “cancer” encompasses bothprimary and metastatic cancers. Representative examples of cancers thatmay be treated using the combination and methods of the inventioninclude, without limitation, carcinoma, lymphoma, blastoma, sarcoma, andleukemia and more particularly bone cancer, gastrointestinal cancer,liver cancer, pancreatic cancer, gastric cancer, colorectal cancer,esophageal cancer, oro-pharyngeal cancer, laryngeal cancer, salivarygland carcinoma, thyroid cancer, lung cancer, cancer of the head orneck, skin cancer, squamous cell cancer, melanoma, uterine cancer,cervical cancer, endometrial carcinoma, vulvar cancer, ovarian cancer,breast cancer, prostate cancer, cancer of the endocrine system, sarcomaof soft tissue, bladder cancer, kidney cancer, glioblastoma and varioustypes of the central nervous system (CNS), etc. The present invention isparticularly useful for treatment of cancers that express PD-L1 (Iwai etal., 2005, Int. Immunol. 17: 133-44), especially metastatic ones andthose that overexpress MUC1 (especially hypoglycosylated form thereof)such as renal cancer (e.g. clear cell carcinoma), prostate cancer (e.g.hormone refractory prostate adenocarcinoma), breast cancer (e.g.metastatic breast cancer), colorectal cancer, lung cancer (e.g.non-small cell lung cancer) liver cancer (e.g. hepatocarcinoma), gastriccancer, bile duct carcinoma, endometrial cancer, pancreatic cancer andovarian cancer. Preferably said cancer is non small cell lung cancer(NSCL).

Representative examples of infectious diseases that may be treated usingthe combination and methods of the invention include, withoutlimitation, a) viral diseases such as those resulting from infection byan herpes virus (HSV1, HSV2, or VZV), a papillomavirus (HPV), a poxviruscausing variola or chicken pox, an enterovirus, a retrovirus such as HIVcausing AIDS, a cytomegalovirus, a flavivirus (e.g. causing Japaneseencephalitis, hepatitis C, dengue and yellow fever), an Hepadnavirus(e.g. HBV), an orthomyxovirus (e.g. influenza virus), a paramyxovirus(e.g. parainfluenzavirus, mumps virus, measles virus and respiratorysyncytial virus (RSV)), a coronavirus (e.g. SARS), rhabdovirus androtavirus; b) diseases resulting from infection by bacteria, forexample, Escherichia, Enterobacter, Salmonella, Staphylococcus,Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus,Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus,Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium,Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella,Yersinia, Haemophilus, or Bordetella; and (c) fungal diseases includingbut not limited to candidiasis, aspergillosis, histoplasmosis,cryptococcal meningitis; and d) parasitic diseases including but notlimited to malaria, Pneumocystis carnii pneumonia, leishmaniasis,cryptosporidiosis, toxoplasmosis, and trypanosome infection.

Typically, upon administration according to the modalities describedherein, the combination product of the invention provides a therapeuticbenefit to the treated subject over the baseline status or over theexpected status if not treated, which can be evidenced by an observableimprovement of the clinical status over the baseline status or over theexpected status if not treated in combination as described herein. Animprovement of the clinical status can be easily assessed by anyrelevant clinical measurement typically used by physicians or otherskilled healthcare staff. In the context of the invention, thetherapeutic benefit can be transient (for one or a couple of monthsafter cessation of administration) or sustained (for several months oryears). As the natural course of clinical status which may varyconsiderably from a subject to another, it is not required that thetherapeutic benefit be observed in each subject treated but in asignificant number of subjects (e.g. statistically significantdifferences between two groups can be determined by any statistical testknown in the art, such as a Tukey parametric test, the Kruskal-Wallistest the U test according to Mann and Whitney, the Student's t-test, theWilcoxon test, etc).

When the method is aimed at treating a proliferative disease, inparticular cancer, a therapeutic benefit can be evidenced by forinstance a reduction in the tumor number; a reduction of the tumor size,a reduction in the number or extent of metastases, an increase in thelength of remission, a stabilization (i.e. not worsening) of the stateof disease, a delay or slowing of disease progression or severity, aprolonged survival, a better response to the standard treatment, animprovement of quality of life, a reduced mortality, etc.

When the method is aimed at treating an infectious disease, atherapeutic benefit can be evidenced by for instance, a decrease of theamount of the infecting pathogenic organism quantified in blood, plasma,or sera of a treated subject, and/or a stabilized (not worsening) stateof the infectious disease (e.g. stabilization of conditions typicallyassociated with the infectious disease such as inflammatory status),and/or the reduction of the level of specific seric markers (e.g.decrease of alanine aminotransferase (ALT) and/or aspartateaminotransferase (AST) associated with liver poor condition usuallyobserved in chronic hepatitis C), decrease in the level of any antigenassociated with the occurrence of an infectious disease and/or theappearance or the modification of the level of antibodies to thepathogenic organism and/or an improved response of the treated subjectto conventional therapies (e.g. antibiotics) and/or a survival extensionas compared to expected survival if not receiving the combinationtreatment.

The appropriate measurements such as blood tests, analysis of biologicalfluids and biopsies as well as medical imaging techniques can be used toassess a clinical benefit. They can be performed before theadministration (baseline) and at various time points during treatmentand after cessation of the treatment. For general guidance, suchmeasurements are evaluated routinely in medical laboratories andhospitals and a large number of kits are available commercially (e.g.immunoassays, quantitative PCR assays).

Preferably, the combination product of the invention is used oradministered for eliciting or stimulating and/or redirecting an immuneresponse in the treated subject. Accordingly, the present invention alsoencompasses a method for eliciting or stimulating and/or re-orienting animmune response (e.g. to tumor or infected cells) comprisingadministering a combination product or composition(s) of therapeuticvaccine and/or one or more immune checkpoint modulator(s) to a subjectin need thereof, in an amount sufficient according to the modalitiesdescribed herein so as to activate the patient's immunity.

In a particular embodiment, the combination product and method(s) of theinvention may be employed according to the modalities described hereinto break immune tolerance usually encountered in chronically infectedand cancerous subjects.

The elicited, stimulated or redirected immune response can be specific(i.e. directed to epitopes/antigens) and/or non-specific (innate),humoral and/or cellular. In the context of the invention, the immuneresponse is preferably a T cell response CD4+ or CD8+-mediated or both,directed to polypeptide(s)/epitope(s), in particular associated with atumor or an infecting pathogenic organism.

The ability of the combination product described herein to elicit,stimulate or redirect an immune response can be evaluated either invitro (e.g. using biological samples collected from the subject) or invivo using a variety of direct or indirect assays which are standard inthe art. For a general description of techniques available to evaluatethe onset and activation of an immune response, see for example Coliganet al. (1992 and 1994, Current Protocols in Immunology; ed J Wiley &Sons Inc, National Institute of Health or subsequent editions). Theability to stimulate a humoral response may be determined by antibodybinding and/or competition in binding (see for example Harlow, 1989,Antibodies, Cold Spring Harbor Press). Evaluation of non-specificimmunity can be performed by for example measurement of the NK/NKT-cells(e.g. representatively and level of activation), as well as, IFN-relatedcytokine and/or chemokine producing cascades, activation of TLRs andother markers of innate immunity (Scott-Algara et al., 2010 PLOS One5(1), e8761; Zhou et al., 2006, Blood 107, 2461-2469; Chan, 2008, Eur.J. Immunol. 38, 2964-2968). Evaluation of cellular immunity can beperformed for example by quantification of cytokine(s) produced byactivated T cells including those derived from CD4+ and CD8+ T-cellsusing routine bioassays (e.g. characterization and/or quantification ofT cells by ELISpot, by multiparameters flow cytometry or ICS, bycytokine profile analysis using multiplex technologies or ELISA), bydetermination of the proliferative capacity of T cells (e.g. T cellproliferation assays by [³H] thymidine incorporation assay), by assayingcytotoxic capacity for antigen-specific T lymphocytes in a sensitizedsubject or by immunization of appropriate animal models. For example,techniques routinely used in laboratories (e.g. flow cytometry,histology) may be used to perform tumor surveillance. One may also usevarious available antibodies so as to identify different immune cellpopulations involved in anti-tumor response that are present in thetreated subjects, such as cytotoxic T cells, activated cytotoxic Tcells, natural killer cells and activated natural killer cells.

If desired, the combination product, composition(s) or methods of theinvention can be used or carried out in association with one or moreconventional therapeutic modalities which are available for treating orpreventing the disease or pathological condition to be treated orprevented (e.g. chemotherapy, radiation, and/or surgery). Suchconventional therapy may be administered to the subject sequentially orconcomitantly with the combination or method according to the invention.

Representative examples of conventional therapeutic drugs that may beuseful in association with the combination product, composition ormethod of the invention include among others nitrosoureas, antibiotics,antimetabolites, antimitotics, antiviral drugs (e.g. interferon alpha),monoclonal antibodies, signaling inhibitors as well as chemotherapeuticdrugs routinely used in cancer therapy. One may cite more specifically:

-   -   alkylating agents such as e.g. mitomycin C, cyclophosphamide,        busulfan, ifosfamide, isosfamide, melphalan, hexamethylmelamine,        thiotepa, chlorambucil, or dacarbazine;    -   antimetabolites such as, e.g. gemcitabine, capecitabine,        5-fluorouracil, cytarabine, 2-fluorodeoxy cytidine,        methotrexate, idatrexate, tomudex or trimetrexate;    -   topoisomerase II inhibitors such as, e.g. doxorubicin,        epirubicin, etoposide, teniposide or mitoxantrone;    -   topoisomerase I inhibitors such as, e.g. irinotecan (CPT-11),        7-ethyl-10-hydroxy-camptothecin (SN-38) or topotecan;    -   antimitotic drugs such as, e.g., paclitaxel, docetaxel,        vinblastine, vincristine or vinorelbine;    -   platinum derivatives such as, e.g., cisplatin, oxaliplatin,        spiroplatinum or carboplatinum;    -   inhibitors of tyrosine kinase receptors such as sunitinib        (Pfizer), sorafenib (Bayer), gefitinib, erlotinib and lapatinib;        and    -   antibodies (in particular anti-neoplastic cetuximab,        panitumumab, zalutumumab, nimotuzumab, matuzumab) or inhibitors        of Human Epidermal Growth Factor Receptor-2 (in particular        trastuzumab); and agents that affect angiogenesis such as, e.g.        inhibitor of Vascular Endothelial Growth Factor (in particular        bevacizumab or ranibizumab);    -   antibiotics conventionally used against infectious pathogenic        organisms such as Aminoglycosides, Ansamycins, Carbapenems,        Cephalosporins, Glycopeptides, Macrolides, Penicillins,        Qionolones and tetracyclins among others; One may cite in        particular antibiotics currently used in first line therapy to        treat a Mtb infection such as isoniazid, rifamycins (e.g.        rifampin, rifapentine and rifabutin), ethambutol, streptomycin,        pyrazinamide and fluoroquinolones as well as those used in        “second-line” therapy in Mtb-infected subjects that has        demonstrated drug resistance such as ofloxacin, ciprofloxacin,        ethionamide, aminosalicylic acid, cycloserine, amikacin,        kanamycin and capreomycin;    -   Antiviral treatment such as interferon-alpha (IFNa and pegylated        derivative) and nucleoside/nucleotide analogues (NUCs). For        example, lamivudine, entecavir, telbivudine, adefovir and        tenofovir are currently used for treating HBV. Other antivirals        include protease inhibitors (e.g. serine protease inhibitors        such as VX950 of Vertex), polymerase inhibitors and helicase        inhibitors that are suitable for treating hepatitis C;    -   TLR agonists and N-glycosylation inhibitors;    -   Interleukins (e.g. IL-2, IL-6, IL-15, IL-24, etc), interferons        (e.g. IFNα, IFNβ or IFNγ), tumor necrosis factor (TNF),        colony-stimulating factors (e.g. GM-CSF, C-CSF, M-CSF, etc) and        chemokines (e.g. CXCL10, CXCL9, CXCL11, etc);    -   siRNA and antisense oligonucleotides that target genes of        infectious pathogenic organism or cellular gene associated with        the targeted disease.

According to an advantageous embodiment, especially when the therapeuticvaccine is armed with a suicide gene, the combination product or methodsaccording to the present invention may be used in association with thecorresponding prodrug (see Table 1). The prodrug is administered inaccordance with standard practice (e.g. per os, systematically, etc).Preferably, the prodrug administration takes place subsequent to theadministration of the therapeutic vaccine, (e.g. at least 3 days afterthe administration of the suicide-gene encoding therapeutic vaccine).The oral route is preferred. It is possible to administer a single doseof prodrug or doses which are repeated for a time which is sufficientlylong to enable the toxic metabolite to be produced within the subject.By way of illustration, a dose of 50 to 500 mg/kg/day, advantageously, adose of 200 mg/kg/day or, preferably, a dose of 100 mg/kg/day isappropriate.

The combination product and method of the invention may also be used incombination with one or more adjuvant(s) or “immune stimulant” toenhance immunity (especially a T cell-mediated immunity), e.g. throughtoll-like receptors (TLR) such as TLR-7, TLR-8 and TLR-9. Forillustrative purposes, such adjuvants include, without limitation, alum,mineral oil emulsion such as, Freunds complete and incomplete (IFA),lipopolysaccharides (Ribi et al., 1986, Immunology andImmunopharmacology of Bacterial Endotoxins, Plenum Publ. Corp., NY,p407-419), saponins such as ISCOMATRIX, AbISCO, QS21 (Sumino et al.,1998, J. Virol. 72: 4931; WO98/56415), imidazo-quinoline compounds suchas Imiquimod (Suader, 2000, J. Am Acad Dermatol. 43:S6), S-27609(Smorlesi, 2005, Gene Ther. 12: 1324) and related compounds such asthose described in WO2007/147529; cationic peptides such as IC-31(Kritsch et al., 2005, J. Chromatogr Anal. Technol. Biomed. Life Sci.822: 263-70), polysaccharides such as Adjuvax and squalenes such asMF59. Other suitable adjuvants include ds RNA like NAB2 with Lipofectin(Claudepierre et al., 2014, J. Virol 88: 5242-55) or 3pRNA (Hornung etal., 2006, Science 314: 994-7), both stimulating IFNα responses viaactivation of cytoplasmic helicase MDA-5 and RIG-1.

Alternatively or in combination, the combination or method according tothe invention can also be used in association with radiotherapy. Thoseskilled in the art can readily formulate appropriate radiation therapyprotocols and parameters (see for example Perez and Brady, 1992,Principles and Practice of Radiation Oncology, 2nd Ed. JB Lippincott Co;using appropriate adaptations and modifications as will be readilyapparent to those skilled in the field). The types of radiation that maybe used in cancer treatment are well known in the art and includeelectron beams, high-energy photons from a linear accelerator or fromradioactive sources such as cobalt or cesium, protons, and neutrons.

The present invention also provides kits including the active entitiesof the combination product of the invention in kit form. A kit is apackaged combination, optionally, including instructions for useoptionally with other components. In one embodiment, a kit includes atleast the therapeutic vaccine as discussed herein in one container andone or more immune checkpoint modulator(s) as described herein inanother container. Such containers are preferably sterile glass orplastic vial. A preferred kit comprises a MVA-based therapeutic vaccine(e.g. a MVA virus expressing the tumor-associated MUC1 antigen and thehuman IL-2) and an immune checkpoint modulator(s) which specificallybinds CTLA-4 (e.g. an anti-CTLA-4 antibody, such as ipilimumab). Anotherpreferred kit comprises a MVA-based therapeutic vaccine (e.g. a MVAvirus expressing the tumor-associated MUC1 antigen and the human IL-2)and an immune checkpoint modulator(s) which specifically binds PD-1(e.g. an anti-PD-1 antibody, such as nivolumab or lanbrolizumab).Another preferred kit comprises a MVA-based therapeutic vaccine (e.g. aMVA virus expressing the tumor-associated MUC1 antigen and the humanIL-2) and an immune checkpoint modulator(s) which specifically bindsPD-L1 (e.g. an anti-PD-L1 antibody, such as MPDL3280A or BMS936559).Optionally, the kit can include suitable devices for performing theadministration of the active agents. The kit can also include a packageinsert including information concerning the compositions or individualcomponent and dosage forms in the kit.

All of the above cited disclosures of patents, publications and databaseentries are specifically incorporated herein by reference in theirentirety to the same extent as if each such individual patent,publication or entry were specifically and individually indicated to beincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the effects on tumor volume (FIG. 1A) and micesurvival (FIG. 1B) in a subcutaneous CT26-CL25 tumor model of theadministration of the formulation buffer (vehicle), MVATG18124 alone,the anti PD-1 clone RMP1.14 alone, a combination of both MVATG18124 andanti PD-1 clone RMP1.14 and a combination of both MVATG18124 and the ratIgG2a isotype control.

FIG. 2 illustrates the effects on mice survival in metastatic CT26-CL25tumor model of the administration of an empty MVA (MVATGN33.1),MVATG18124 alone, an anti CTLA-4 antibody alone, its IgG2b isotypecontrol, a combination of both MVATG18124 and anti CTLA-4 and acombination of both MVATG18124 and the IgG2b isotype control.

FIG. 3A illustrates the percentage of gated CD8⁺CD3^(dim) cells(referred as CD8^(dim)CD3^(dim) herein after) in dissociated lungsobtained from untreated (i.e. naïve) mice or mice treated withMVATG18124, anti-CTLA-4 and both MVATG18124 and anti-CTLA-4. FIG. 3Billustrates an example of CD8^(dim)CD3^(dim) (referred as CD8⁺CD3^(dim)in the Figure) population in CD3/CD8 dot blot lung cells from micetreated with anti CTLA-4 and MVATG18124.

FIG. 4 represents the IFN-γ positive CD8^(dim)CD3^(dim) cell populationobtained following ConA induction in lung samples obtained fromuntreated (i.e. naïve) mice or mice treated with MVATG18124, anti-CTLA-4and both. FIG. 4A: ConA-induced fold induction of percentage of IFNγ⁺CD8^(dim)CD3^(dim) cells upon incubation of lung cells with bmDCs tofacilitate activation. FIG. 4B: ConA-induced fold induction ofpercentage of IFNγ⁺ CD8^(dim)CD3^(dim) cells upon incubation of lungcells with anti-CD28 to facilitate activation.

FIG. 5 illustrates ELIspot analysis of splenic lymphocytes stimulatedwith the β-gal-specific peptide T9L3 showed β-gal specific response insplenic lymphocytes treated with MVATG18124. The raw data wastransformed into histogram graph. Results are expressed as number ofspot forming units (sfu) per 1×10⁶ splenocytes (mean) for eachtriplicate or quadriplicate.

FIG. 6 represents the induction of IFN-γ, CD107a and KLRG1 positivecells in CD8^(dim)CD3^(dim) cell population obtained in lung samplesstimulated with T9L-3 peptide (hatched) or irrelevant T8G peptide(dotted) following treatment with an empty vector MVATGN33.1 alone or incombination with anti-CTLA-4, MVATG18124 alone or in combination withanti-CTLA-4 or anti-CTLA-4 alone.

FIG. 7 illustrates the effect provided by TG4010 treatment on micesurvival and tumor volume in a CT26-MUC1 tumor model. FIG. 7A: CT26-MUC1lung tumor model. CT26-MUC1 cells were injected iv. Day 2 and 9, 5.10⁷pfu TG4010, empty MVA vector (MVATGN33.1) or buffer (SO8) were injectedintravenously. Mice were weighed twice per week and sacrificed whenloosing 10% of weight. Percent survival was monitored. FIG. 7B:CT26-MUC1 s.c. tumor model. CT26-MUC1 cells were injected s.c. Day 2 and9, 1.10⁷ pfu TG4010, empty MVA vector (MVATGN33.1) or buffer (SO8) wereinjected s.c. in the same flank. Mice were sacrificed when the tumorsreached the size of 2000 mm³. Mean tumor volumes with SEM are shown.

FIG. 8 illustrates the effect on mice survival (FIG. 8A) and tumorvolume (FIG. 8B) provided by TG4010 treatment in combination with antiPD-1 antibody in the CT26-MUC1 s.c. tumor model. BALB/c mice wereinjected s.c. with 2.10⁵ CT26-MUC1 cells. On days 2 and 9 after tumorimplantation, mice were treated sc with TG4010 (also called MVATG9931)or an empty control vector at the suboptimal dose of 1.10⁶ pfu followedby i.p. administration of 250 μg anti PD-1 (RMP1.14, IgG2a, BioXCell) ondays 10, 13, 15 and 17. Mice were sacrificed when the tumors reached thesize of 2000 mm³. Percent survival and mean tumor volumes were monitoredover time.

FIG. 9 illustrates the survival of BALB/C mice injected s.c. withCT26-MUC1 tumor cells and immunized with two injections of 1×10⁷ PFU ofMVATGN33.1 or MVATG9931 on days D2 and D9 followed by i.p.administrations of 250 μg of anti-PD-1 Ab on days D10, D13, D15 and D17.(*: p<0.05; **:: p<0.01; ***: p<0.001)

EXAMPLES

We set out to combine immune checkpoint blocking approaches withtherapeutic MVA vectors with the goal of inducing antigen-specific Tcell immune response with MVA and release the brakes from T cellgeneration with immune checkpoint antibodies. Preclinical evidence forsynergistic effects of immune checkpoint blockers combined with viralvectors was to be demonstrated in mouse tumor models. This implies theuse of i) murine-specific anti-immune check point antibodies and ii) anantigen-expressing MVA vector.

The MVA vector chosen for these studies (MVATG18124) contains thebacterial LacZ gene encoding the beta-galactosidase Ag model (SEQ ID NO:3) under the control of poxvirus promoter pH5R (SEQ ID NO: 4). pH5Rpromoter was isolated by PCR amplification from Vaccinia virusCopenhagen strain using appropriate primers. The E. Coli LacZ gene wasobtained by PCR amplification using primers otg19678 (SEQ ID NO: 5) andotg19679 (SEQ ID NO: 6) with pCMVBeta (Clontech) as DNA template. ThepH5R and LacZ genes were cloned into a shuttle plasmid between MVAsequences extending from positions 142006 to 142987 and positions 142992to 143992 according to GenBank sequence EF675191.1. MVATG18124 wasgenerated into chicken embryo fibroblast (CEF) cells by transfection ofshuttle plasmid into previously MVA-infected CEF, resulting inhomologous recombination between shuttle plasmid DNA and MVA genome andinsertion of the pH5R-LacZ cassette into deletion III. Recombinant MVAclones were isolated using conventional technology (Lullo et al., 2010,J Virol Methods 163: 195-204) and the selected clones were controlled byPCR, then amplified in CEF cells. Virus stocks were titrated on DF1cells by plaque assay. Absence of mutation into the inserted DNA and thesurrounding region was checked by DNA sequencing.

It was first chosen to target the immune checkpoint blocker murine PD-1(mPD-1) with an appropriate antibody. The rat anti mPD-1 antibodyRMP1-14 (BioXcell) was chosen. This antibody was shown to block theinteraction of mPD1 with its ligands (Yamazaki et al., 2005, J. Immunol.175(3): 1586-92).

The combination of mPD-1 inhibitors with the antigen-expressingMVATG18124 was tested in vivo in two mice models, respectivelymetastatic and subcutaneous tumor models. The colon carcinoma cell lineCT26.CL25 (ATCC CRL-2639), transduced with the LacZ gene and thus stablyexpressing beta-galactosidase, was either injected subcutaneously togenerated palpable tumors (subcutaneous model) or intravenously togenerate lung metastasis. Mice were then treated with MVATG18124expressing beta-galactosidase and murine-specific immune checkpointblockers like anti PD-1 or anti CTLA-4 antibodies.

Example 1: Combination of MVATG18124 with Anti-PD-1 Mab

The combination of mPD-1 inhibitor (commercial clone RMP1-14; BioXCell)with beta-gal-expressing MVATG18124 was tested in vivo in a subcutaneoustumor model. Balb/c mice were subcutaneously injected with 2×10⁵CT26.CL25 cells. Day 2 and 9 after cell implantation, mice were thenintravenously immunized with either 1×10⁴ pfu of MVATG18124 orformulation vehicle as negative control in combination with 4intraperitoneal (ip) administrations at days 10, 13, 15 and 17 of 250 μgof either murine anti PD-1 antibody RMP1.14 (BioXcell) or its isotypecontrol IgG2a (clone 2A3). In other terms, 5 groups of 10 mice weretested; a first group treated with MVATG18124 receiving 2 iv injections(group 1); a second group treated with 4 ip injections of anti-PD-1antibody (group 2); a third group treated with both MVATG18124 andanti-PD-1 antibody receiving 2 iv injections of the therapeutic MVA and4 ip injections of anti-PD-1 antibody (group 3); a fourth groupreceiving 2 iv injections of the therapeutic MVATG18124 and 4 ipinjections of isotype antibody (group 4) and a control group receivingthe formulation buffer (group 5). Tumor growth and survival weremeasured over time as illustrated in FIG. 1.

As expected, tumor volume increased very rapidly in control groupreceiving formulation buffer. Rapid tumour growth was also observed inthe group receiving mPD-1 antibody. A slight delay in tumor volume wasseen in groups receiving MVATG18124 either alone or in combination withthe isotype control. Tumor growth was greatly reduced in the“combination” group injected with both MVATG18124 and mPD-1 antibodies(FIG. 1A). Effects on mice survival are more pronounced since about 70%of mice treated with both MVATG18124 and mPD-1 antibodies are stillalive more than 100 days following tumor implantation versus 10% ofMVATG18124-treated animal group (significant differences). In contrast,control, antibody-treated and isotype-treated animals died within lessthan 50 days (FIG. 1B).

Example 2: Combination of MVATG18124 with Anti-CTLA-4 Mab

The combination of CTLA-4 inhibitor (commercial clone 9D9; BioXCell)with antigen-expressing MVATG18124 was tested in vivo in a metastaticCT26-CL25 model. 2×10⁵ CT26.CL25 cells were injected intravenously (iv)in Balb/c mice. Days 2 and 9, MVATG18124 encoding beta-galactosidase orits empty vector control MVAN33.1 was administered iv at the dose of1.10⁴ pfu. 250 μg of the murine anti-CTLA4 antibody 9D9 (BioXCell) orits IgG2b isotype control (clone MPC-11 BioXCell) were injectedintraperitoneally (ip) at days 3 and 10. The survival of mice wasfollowed for more than 60 days. The viral dose of 1.10⁴ pfu wasidentified as optimal dose to increase survival rates in this tumormodel (data not shown).

As illustrated in FIG. 2, treatment with anti CTLA4 antibodies or itsisotype control alone showed weak effects comparable to those observedwith the empty MVA vector MVATGN33.1 (35% survival at day 20 for allthree groups). In contrast, treatment with MVATG18124 alone or incombination with the isotype control increased mice survival (35%survival at day 35 for both groups). The group of mice treated with thecombination of MVATG18124 and anti-CTLA4 antibodies greatly increasedthe percentage of 35% survival to more than 60 days.

Thus, we have clearly demonstrated a clear anti-tumor effect of treatingMVA-based immunotherapeutic vaccine and the immune checkpoint blockeranti CTLA4.

Example 3: Lymphoid Cell Population Studies in Dissociated Lungs ofTreated Mice Determination of IFNγ Positive CD8dim CD3dim Cells

Cellular response was examined in mice treated with either theanti-CTLA-4 antibody the antigen-expressing MVA or both as well as inuntreated (i.e. naïve) mice. Five BALB/c mice per group were injected ivwith MVATG18124 (1.10⁴ pfu) day 1 and 8 or ip with 250 μg anti CTLA-4(clone 9D9, BioXCell) day 2 and 9 or both. Mice were sacrificed day 15,and lungs were isolated. Lungs from all 5 mice per group were pooled,cut into small pieces in C-tubes (Miltenyi) and enzymaticallydissociated using a tissue dissociation kit (Miltenyi, 130-096-730)using the Gentle OctoMACS (Miltenyi) according to the manufacturer'srecommendations.

Lung-derived cells were plated at 2.10⁶ cells/well (96 well plate) in Tcell-specific medium (TexMACS, Miltenyi). Cells were activated byco-cultivation with peptide-loaded bone marrow-derived murine dendriticcells (bmDCs): bmDCs from BALB/c mice were generated from bone marrowcells matured in the presence of murine GM-CSF (Peprotech, 100 μg/ml)for 10 days. Alternatively, activation was facilitated by incubationwith 1 μg of anti CD28 (clone PV-1). Concanavalin A (ConA, 5 μg/ml)served as non-specific activator. Anti CD107a antibody (clone eBio1D4B)was added to label degranulating cells. Secretion of cytokines wasblocked after one 20 hour of incubation by adding GolgiPlug/Brefeldin(1:1000, BD Biosciences). After 5 hours total incubation time, cellswere washed and stained for viability (LiveDead, Fixable violet deadcell staining kit) and surface markers CD8a (clone 53-6.7) and CD3c(clone 145-2C11). Cells were stained intracellularly for IFN-γ (cloneXMG1.2) using the BD Cytofix/Cytoperm kit (BD Biosciences). Cells werefixed and analyzed by flow cytometry (Navios, Beckman Coulter).

Combinatorial treatment of MVATG18124 and anti CTLA-4 in naïve BALB/cmice leads to the appearance of a sub population of lymphocyte cellsnamed CD8^(dim)CD3^(dim) in the lung. FIG. 3B represents an example ofCD8^(dim)CD3^(dim) population in CD3/CD8 dot blot lung cells from micetreated with anti CTLA-4 and MVATG18124. Gating living lymphocytes, thepercentages of gated CD8^(dim)CD3^(dim) cells in two independentexperiments are illustrated in FIG. 3A. More specifically, thepopulation of CD8^(dim)CD3^(dim) was clearly increased after treatmentwith MVATG18124 and even more after combinatorial treatment withMVATG18124+anti CTLA-4.

Next, the percentage of intracellular IFN γ⁺ cells was assessed in theCD8^(dim)CD3^(dim) population. Within this CD8^(dim)CD3^(dim)population, highest induction of IFN-γ+ cells was observed in micetreated with MVATG18124+anti CTLA-4 (FIGS. 4A and 4B).

In summary, combinatorial treatment of MVATG18124 and anti CTLA-4 innaïve BALB/c mice leads to the appearance of a CD8^(dim)CD3^(dim)lymphocyte cell population in the lung. Upon stimulation with ConA and ahigh percentage of CD8^(dim)CD3^(dim) from mice treated withMVATG18124+anti CTLA-4 can be induced to secrete IFN-γ.

The CD3^(dim)CD8^(dim) cell population was analyzed in greater detail.

-   -   In the course of our analyses, we observed that the        CD3^(dim)CD8^(dim) population was positive for the killer cell        lectin like receptor G1 (KLRG1′) and negative or low for CD127        (IL-7Rα) (CD127^(−/low)). This phenotype is associated with        antigen experienced short lived effector cells (SLECs) (Obar et        al., 2011, J Immunol., doi: 10.4049/jimmunol.1102335; Sarkar et        al., 2008, J Exp Med 205(3): 625-40).    -   The CD3^(dim)CD8^(dim) KLRG1⁺ population infiltrating/present in        the lung of mice treated with MVATG18124 and anti CTLA-4        responds to an antigen-specific stimulus with IFN-γ secretion        and degranulation (CD107a).        Determination of IFN-γ, CD107a and KLRG1 Positive Cells in        CD8^(dim)CD3^(dim) Cell Population

As described above, BALB/c mice were injected i.v. with MVA-β-gal or anempty vector MVATGN33.1 at 1.10⁴ pfu. On days 3 and 10, mice received250 μg anti CTLA-4 i.p. Lungs were taken day 14 and enzymaticallydissociated. Lung derived cells were plated at 2.10⁶ cells/well (96 wellplate) in T cell-specific medium (TexMACS, Miltenyi), activated byincubation with 1 μg of anti CD28 (clone PV-1) and stimulated with aβ-gal specific peptide (T9L-3) or a control peptide (T8G) in thepresence of anti CD107a antibody (clone eBio1D4B) to label degranulatingcells. Secretion of cytokines was blocked after one hour of incubationby adding GolgiPlug/Brefeldin (1:1000, BD Biosciences). After 5h totalincubation time, cells were washed and stained for viability (LiveDead,Fixable violet dead cell staining kit) and for the surface markers CD8a(clone 53-6.7), CD3ε (clone 145-2C11) and KLRG1 (clone 2F1/KLRG1). Cellswere stained intracellularly for IFN-γ (clone XMG1.2) using the BDCytofix/Cytoperm kit (BD Biosciences). Cells were fixed and analyzed byflow cytometry (Navios, Beckman Coulter). After 5 hours, cells andstained CD8a.

Treatment with MVATG18124 or an empty control vector, and even more thecombination of MVATG18124 and anti CTLA-4 in naïve BALB/c mice lead tothe appearance of a CD8^(dim)CD3^(dim)KLRG1⁺ lymphocyte cell populationin the lung. Upon ex vivo stimulation with the β-gal peptide T9L-3, thispopulation secreted IFNγ and degranulated (CD107a) and, as illustratedin FIG. 6, the percentage was higher after treatment with MVATG18124 andanti CTLA-4 than in the other groups treated with the control vector(MVATGN33.1 alone or with anti CTLA-4), with MVATG18124 alone or withanti CTLA-4 alone. As expected, stimulation with an irrelevant peptide(T8G peptide) did not yield such induction.

In conclusion, the treatment with MVATG18124 and anti CTLA-4 increasesthe b-gal specific response in a CD3^(dim)CD8^(dim)KLRG1⁺ cellpopulation in the lung.

Example 4: Secretion of IFN-γ

Further, the number of IFNγ secreting splenic lymphocytes wasinvestigated following BALB/c mice treatment with MVATG18124 or MVAN33.1at 1.10⁴ pfu (D1 and 8) and anti CTLA-4 or its isotype control (D2 andD9, 250 μg ip). Measurement was performed at day 14 by ELISpot(Enzyme-linked immunospot) assay.

Plate Preparation

The day preceeding the experiments, membrane ELISpot plates (Millipore,ref. MSIPS4W10) were prewetted with 15 μL of 35% ethanol per well withmaximum incubation time of 2 min. Plates were washed five times with 200μL per well of sterile water.

ELISpot plates were coated with a rat anti-mouse IFN-γ monoclonalantibody (AN18 Mabtech, ref. 3321-3-1000) diluted at 15 μg/mL in sterileDPBS (Sigma, ref. D8357) (100 μL/well). The plates were covered andincubated overnight at 4° C. The next day, the plates were washed 3times with sterile PBS (200 μL/well) and were saturated for 1 h at 37°C. with 200 μL/well of complete RPMI 1640 medium (RPM11640 medium,(Sigma R0883); L-Glutamine 2 mM, (Sigma G6392); Gentamycin 0.01 g/L(Schering Plough U570036); Fetal Calf Serum 10% (JRH 12003-1000M) 550 μlof a solution 5×10⁻² M bmercaptoethanol).

Sample Preparation

For ex vivo evaluation of the frequency of the specific CD8+ T cellsinduced by immunization, euthanized animals were splenectomized 7 daysafter last immunization. Spleens from the same group were pooled in acell strainer in a well of a 6-wells culture plate containing 5 mL ofcomplete medium. Spleens were crushed with a syringe piston and the cellstrainer discarded. Splenocytes were collected with 8 mL of completemedium and transferred in a 15 mL falcon tube. The splenocytessuspension was laid over 4 mL of Lympholyte®-M separation cell media(Cedarlane, ref. CL5035) and centrifuged (20 min, 1500×g, roomtemperature). The interphase containing lymphocytes was collected andrinsed three times with 10 mL of PBS. Between each rinse step, cellswere centrifuged (4 min at 400 xg) and the supernatants were discarded.The remaining red blood cells were lysed by addition on the lymphocytepellet of 2 mL of RBC lysis buffer 1× (10× solution: BD Pharm Lyse™lysing solution, ref. 555899) diluted in sterile water. Each tube wasgently vortexed immediately after adding the lysis solution andincubated at room temperature for 15 minutes. Lysis was topped by theaddition of 10 mL DPBS followed by centrifugation 4 min at 400×g., thecells were resuspended in 10 mL of complete RPMI 1640 medium. The cellswere counted with a Z2 Cell Counter (Beckman Coulter) and the cellconcentration was adjusted at 1×10⁷ cells per mL in complete RPMI 1640medium.

Assay

To perform the ELISpot assay, 100 μL of lymphocyte suspension from eachgroup (1×10⁶ cells) were added to each wells of a coated 96-wells plate.One given condition was tested in triplicates or in quadriplicates. Onehundred microliter of different indicated peptides (2 μg/mL in completeRPMI 1640 medium) was added to the cell suspension. ConA (Sigma, ref.C5275) was used as positive control (5 μg/mL final concentration)MVA-specific peptide (S9L-8) was used for immunization control. Theplates were then incubated at 37° C. in 5% CO₂ for 16 to 20 hours. Then,plates were washed three times with DPBS (200 μL). Biotinylated ratanti-mouse IFN-γ monoclonal antibody (Mabtech, ref. 3321-6-1000) wasdiluted at 1 μg/mL in antibody mix buffer (PBS, 0.5% SVF) anddistributed at 100 μL/well. Plates were incubated 2 hours at roomtemperature in darkness, and then washed three times in DPBS (200 μL).One hundred microliter of Extravidin-Phosphatase alkaline (SIGMA, ref.E2636) (Diluted 1/5000 in antibody mix buffer) was added to each welland the plates were incubated for 1 hour at room temperature indarkness. Plates were finally washed three times in DPBS (200 μL). Onehundred microliter of BCIP/NBT (Sigma, ref. B5655, one caps in 10 mLMilliQ water) was added to each well until blue spots develop and thenplates were washed thoroughly in tap water and dried.

Data Acquisition

Spots were counted with an ELISpot reader (CTL Immunospot reader, S5UV). A visual quality control (comparing machine scans and plates) wasperformed on each well to ensure that the counts provided by the ELISpotreader match the reality of the picture. Results were expressed asnumber of spot forming units (sfu) per 1×10⁶ splenic lymphocytes (mean)for each triplicate or quadriplicate. Specific ELISpot response wasdetermined either with the DFR(eq) method (Moodie et al., Cancer ImmunolImmunother. 2010 October; 59(10):1489-501) or with an empirical cut-offcalculated as the mean number of spots from blank wells plus two timesthe standard deviation of this mean number of spots.

As shown in FIG. 5, ELIspot analysis of splenic lymphocytes stimulatedwith the β-gal-specific peptide T9L3 showed β-gal specific response insplenic lymphocytes treated with MVATG18124. The specific responsesincreased further after combinatorial treatment with anti CTLA-4 but notwith its isotype control. Stimulation with the non-specific peptide T8Gshowed no increase of IFN-β secreting cells.

Example 5: Combinatorial Effect of TG4010 (MVA-MUC1-IL-2) and Anti PD-1(RMP1.14) in a MU1-Positive CT26-Based Tumor Model

TG4010 is a MVA vector encoding the full cDNA sequence of human MUC1 andhuman IL-2. Anti-tumoral efficacy provided by this vector was tested ina CT26-based MUC1-positive cell line which gives rise to MUC1-positivetumors after s.c. injection, as well as lung tumors after i.v.injection.

Generation of CT26-MUC1 Cell Line

The murine colon carcinoma cell line CT26 WT (ATCC CRL-2638) was stablytransfected with the plasmid pTG5077 encoding the full cDNA sequence ofhuman MUC1 under the control of the CMV promoter as well as aG418-resistance gene under the control of the SV40 promoter. CT26 cellswere transfected by means of Lipofectamine LTX with pTG5077, andcultivated in the presence of 0.4 mg/ml G418 to select for stabletransfectants. After 14 days, living cells were labeled with amonoclonal antibody against MUC1 (H23+second antibody Goat antimouse-FITC). Positive cells were sorted (FACS ARIA), transferred in 96well plates at 1 cell/well. Outgrowing clones were analyzed for stableMUC1 expression by flow cytometry up to day 60 after transfection. Fourstably MUC1-expressing clones were then tested for their ability toinduce tumor growth in BALB/c mice after sc injection and after ivinjection. One clone was retained after verification that s.c.-implantedtumors and lung tumors obtained after iv injection were MUC1-positive.

Therapeutic Efficacy of TG4010 in the CT26-MUC1 Tumor Model

2.10⁵ CT26-MUC1 cells were injected s.c. or i.v. in BALB/c mice togenerate sc tumors or lung tumors, respectively. On day 2 and 9 aftertumor challenge, mice were treated s.c. or i.v., respectively, with1.10⁷ TG4010 or the empty control vector MVATGN33.1. Mean tumor volumeor percent survival were monitored over time. TG4010 showed significantimprovement of survival in the iv/iv lung tumor model (p=0.00642) andsignificant reduction of tumor growth in the sc/sc tumor model (seeFIGS. 7A and 7B, respectively).

Therapeutic Efficacy of TG4010 in Combination with Anti-PD1 in theCT26-MUC1 Tumor Model

BALB/c mice were injected s.c. with 2.10⁶ CT26.MUC1 cells. On days 2 and9 after tumor implantation, mice were treated sc with TG4010 (alsodesignated MVATG9931) or an empty control vector (MVATGN33.1) at thesuboptimal dose of 1.10⁶ pfu. On days 10, 13, 15 and 17, mice received250 μg anti PD-1 (RMP1.14, IgG2a, BioXCell). Mice were sacrificed whenthe tumors reached the size of 2,000 mm³. Tumor volume and animalsurvival were followed over time.

As illustrated in FIG. 8B, tumors started to grow at day 16 and tumorvolume increased rapidly and regularly over time in control groupreceiving formulation buffer, as expected. A delay in tumour growth wasobserved in the groups receiving mPD-1 antibody, TG4010 alone and emptyMVATGN33.1 alone or with anti-PD-1. In contrast, tumor growth wasgreatly reduced in the “combination” group injected with both TG4010 andmPD-1 antibody. Effects on mice survival are also observed as shown inFIG. 8A. Indeed, about 70% of mice treated with both TG4010 and mPD-1antibody are still alive more than 60 days following tumor implantationwhile approximately 10% and 20% of animals treated with the emptycontrol (without or with the anti-PD-1 antibody) remained alive. Only 30and 40% of animals respectively treated with TG4010 alone or withanti-PD-1 antibody survived 2 months after tumor implantation. Incontrast, control group treated with formulation buffer died within lessthan 45 days. The increased survival provided by the combinatorialtreatment was maintained overtime (120 days after tumor implantation)(data not shown).

The same experiment as above was also conducted except that mice weretreated with 2 injections of 1.10⁷ pfu of MVATG9931 (i.e. TG4010) or thenegative MVATGN33.1 control vector, optionally followed by four i.p.administrations of 250 μg of anti-PD1. As illustrated in FIG. 9, asubstantial increase in median survival of 70 days for the combinatorialtreatment is however noticeable in comparison to 46 and 49.5 days forthe mice treated with MVATG9931 and anti-PD-1, respectively. Note alsothat the survival increase is significant (p=0.008) when the combinationof MVATG9931 and anti-PD-1 treatments is compared to the control emptyvirus MVATGN33.1 and anti-PD-1 modalities, suggesting a MUC-1 specificinteraction at this dose of virus. At the highest dose of 1×10⁷ PFU forMVATG9931, the best therapeutic activity among all the conditions testedis achieved for the combination with significance reached against eachtreatment modality alone (p=0.014 against MVATG9931 and p<0.001 againstanti-PD-1; FIG. 9). In conclusion, the combination of MVATG9931 with ananti-PD-1 antibody allowed to obtain the best therapeutic index in theectopic CT26-MUC1 model in comparison to each treatment alone whichpaves the way to the clinical evaluation of this combination therapyapproach.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific method and reagents described herein, including alternatives,variants, additions, deletions, modifications and substitutions. Suchequivalents are considered to be within the scope of this invention andare covered by the following claims.

1. A combination product comprising at least (i) a therapeutic vaccineand (ii) one or more immune checkpoint modulator(s).
 2. The combinationproduct of claim 1, wherein said therapeutic vaccine comprises arecombinant plasmid or a viral vector.
 3. The combination product ofclaim 2, wherein said therapeutic vaccine comprises a poxvirus.
 4. Thecombination product of claim 2, wherein said therapeutic vaccinecomprises a human or an animal adenovirus.
 5. The combination product ofclaim 4, wherein said adenovirus is replication-defective.
 6. Thecombination product of claim 1, wherein said therapeutic vaccinecomprises or encodes one or more polypeptide(s) selected from the groupconsisting of suicide gene products, immunostimulatory polypeptides, andantigenic polypeptides.
 7. The combination product of claim 6, whereinsaid therapeutic vaccine comprises or encodes one or more polypeptide(s)selected from the group consisting of the MUC1 antigen, HPV E6 and E7antigens, the human IL-2, the human GM-CSF, the FCU1 suicide gene, HBVantigens, mycobacteria antigens, and the HCV non structural antigens. 8.The combination product of claim 7, wherein said therapeutic vaccinecomprises a MVA virus encoding the MUC-1 antigen and IL-2; a MVA virusencoding a fusion of NS3 and NS4 HCV antigens and NS5b antigen; a MVAvirus encoding membrane anchored HPV-16 non-oncogenic E6 and E7 antigensand IL-2; a MVA virus encoding the FCU1 suicide gene; a MVA virusencoding a combination of TB antigens or an Ad vector encoding acombination of HBV antigens.
 9. (canceled)
 10. The combination productof claim 1, wherein said one or more immune checkpoint modulator(s) is ahuman or humanized monoclonal antibody that specifically binds to any ofPD-1, PD-L1, PD-L2, LAG3, Tim3, BTLA, SLAM, 2B4, CD160, KLRG-1, andCTLA4.
 11. The combination product of claim 10, wherein at least one ofsaid one or more immune checkpoint modulator(s) is selected from thegroup consisting of (a) an antibody that specifically binds to humanPD-1; (b) an antibody that specifically binds to human PD-L1; and (c) anantibody that specifically binds to human CTLA-4.
 12. The combinationproduct of claim 1, wherein said one or more immune checkpointmodulator(s) is/are expressed by said therapeutic vaccine.
 13. Thecombination product of claim 1, wherein said combination product is acomposition comprising a therapeutically effective amount of at leastsaid therapeutic vaccine and said one or more immune checkpointmodulator and a pharmaceutically acceptable vehicle.
 14. The combinationproduct of claim 1, comprising from about 0.5 mg/kg to about 25 mg/kg ofthe immune checkpoint modulator(s).
 15. The combination product of claim1, wherein said combination comprises from approximately 10⁸ vp toapproximately 5×10¹¹ vp of an adenoviral vector or from approximately10⁶ pfu to approximately 10¹⁰ pfu of a MVA vector.
 16. The combinationproduct of claim 15, wherein the immune checkpoint modulator(s)comprised in said combination is formulated for intravenous orintratumoral administration and wherein the therapeutic vaccinecomprised in said combination is formulated for intravenous,intramuscular, subcutaneous or intratumoral administration.
 17. Thecombination product of claim 1, wherein the administrations of saidtherapeutic vaccine and said one or more immune checkpoint inhibitor isconcomitant, sequential, or interspersed.
 18. The combination product ofclaim 17, wherein the administration(s) of said therapeutic vaccinestarts before the administration(s) of said immune checkpointmodulator(s).
 19. The combination product according to claim 1comprising from 4 to 15 administrations of 10⁷ or 10⁹ pfu of a MVA-basedtherapeutic vaccine at approximately 1 to 3 week interval interspersedwith 2 to 6 administrations of 3 to 10 mg/kg of anti-immune checkpointantibody/antibodies every 2 or 3 weeks.
 20. A composition comprisingeffective amounts of: (a) a therapeutic vaccine that comprises arecombinant plasmid or viral vector; and (b) one or more immunecheckpoint modulator(s) that is/are a human or humanized monoclonalantibody/antibodies that specifically bind(s) to any of PD-1, PD-L1,PD-L2, LAG3, Tim3, BTLA, SLAM, 2B4, CD160, KLRG-1, and CTLA4.
 21. Amethod for treating a proliferative or an infectious disease comprisingadministering: (a) a combination product comprising at least (i) atherapeutic vaccine and (ii) one or more immune checkpoint modulator(s);or (b) a composition comprising effective amounts of: (1) a therapeuticvaccine that comprises a recombinant plasmid or viral vector; and (2)one or more immune checkpoint modulator(s) that is/are a human orhumanized monoclonal antibody/antibodies that specifically bind(s) toany of PD-1, PD-L1, PD-L2, LAG3, Tim3, BTLA, SLAM, 2B4, CD160, KLRG-1,and CTLA4.
 22. The method of claim 21, wherein said proliferativedisease is a cancer and wherein said infectious disease results frominfection with a virus selected from the group consisting of herpesvirus papillomavirus, poxvirus, retrovirus, HCV, HBV, and influenzavirus.
 23. A method for eliciting or stimulating and/or re-orienting animmune response comprising administering: (a) a combination productcomprising at least (i) a therapeutic vaccine and (ii) one or moreimmune checkpoint modulator(s); or (b) a composition comprisingeffective amounts of: (1) a therapeutic vaccine that comprises arecombinant plasmid or viral vector; and (2) one or more immunecheckpoint modulator(s) that is/are a human or humanized monoclonalantibody/antibodies that specifically bind(s) to any of PD-1, PD-L1,PD-L2, LAG3, Tim3, BTLA, SLAM, 2B4, CD160, KLRG-1, and CTLA4 to asubject in need thereof so as to activate the patient's immunity. 24.The method of claim 21 which is used or carried out in association withone or more conventional therapeutic modalities.
 25. A kit comprising:(a) a container comprising a therapeutic vaccine that comprises arecombinant plasmid or viral vector; and (b) a different containercomprising one or more immune checkpoint modulator(s) that is/are ahuman or humanized monoclonal antibody/antibodies that specificallybind(s) to any of PD-1, PD-L1, PD-L2, LAG3, Tim3, BTLA, SLAM, 2B4,CD160, KLRG-1, and CTLA4.
 26. The combination product of claim 3,wherein said poxvirus is a vaccinia virus.
 27. The method of claim 23which is used or carried out in association with one or moreconventional therapeutic modalities